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The RNA exosome is an essential 3′ to 5′ exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3′ to 5′ exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.

Background Chronic hepatitis B-related liver cirrhosis(HBV-LC)is the most common cirrhosis in China, which is characterized as liver damage and high mortality. We aim to investigate the characteristics of TRAIL+NK cells in patients with HBV-LC and their relationship with liver damage in patients with HBV-LC. Methods Thirty cases each of chronic hepatitis B (CHB), HBV-related compensated liver cirrhosis (HBV-CLC) and HBV-related decompensated liver cirrhosis (HBV-DLC) patients were recruited in this study. Thirty age‐and sex‐matched healthy individuals were recruited as healthy controls (HCs). NK cell phenotypes were determined using flow cytometry. Serum chemokine concentrations were ascertained using the CBA Flex set. Cell apoptosis was analyzed using the Annexin V-PE/7-AAD apoptosis Kit. Results CD56bright NK cells increased, but CD56dim NK cells reduced in HBV-LC patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was mainly expressed on CD56bright NK cells. As the degree of liver damage increased, the frequency and activation of total TRAIL+NK cells and TRAIL+NK cell subsets continued to increase, especially in the HBV-LC patients. Furthermore, the difference in frequency and activation of total TRAIL+NK cells between the HBV-CLC and HBV-DLC groups was mainly due to the highly activation and increase of TRAIL+CD56bright NK cells. With the increasing degree of liver damage, CXCR3-associated chemokines (including CXCL9, CXCL10 and CXCL11) were constantly increased, particularly in the HBV-DLC group. The expression of CXCR3 on CD56bright NK cells was almost 100 % in all enrolled cohorts. CXCR3-associated chemokines were negatively correlated with liver function and positively correlated with fibrosis degree. TRAIL+CD56bright NK cells were negatively correlated with liver function, and positively correlated with fibrosis degree and CXCR3-associated chemokines. The apoptosis of K562 cells and hepatocytes was suppressed partially by the TRAIL-neutralizing antibodies. Conclusions The increase of CXCR3-related chemokines (including CXCL9, CXCL10 and CXCL11) might be related to the migration of TRAIL+ CD56bright NK cells to the liver. Highly activated TRAIL+ CD56bright NK cells were associated with the liver damage in HBV-LC patients. These findings may provide new perspectives and theoretical basis for future immunotherapy of HBV-LC patients.

Developing K-12 students’ computational thinking (CT) skills is essential. Building on the existing literature that has emphasized programming skill development, this study expands the focus to examine students’ use of underlying CT cognitive skills during collaborative problem-solving processes. A case study approach was employed to examine video data of 5th graders engaging in an integrated-STEM robotics curriculum. The findings reveal that students applied algorithmic thinking most frequently and prediction the least. They recorded most debugging behaviors initially in the problem-solving process, but after accumulating more experiences their uses of other CT skills, including algorithmic thinking, pattern recognition, and prediction, increased. Implications for developing young learners’ CT skills to solve real-world problems are discussed.

NK cells play an important role in immune regulation and defense of infection, but their characteristics in patients with decompensated cirrhosis and their relationship with liver function remain unclear. We studied the functional properties of NK cells (including CD56dim NK and CD56bright NK cells) in patients with HBV-related decompensated liver cirrhosis (HBV-DLC) and analyzed their relationship with decompensation of liver function. Thirty patients with HBV-DLC and 25 patients with HBV-related compensated liver cirrhosis (HBV-CLC) were recruited in this study. Twenty five age- and sex-matched healthy individuals were recruited as healthy controls (HCs). The phenotypical and functional characteristics of NK cell subsets were detected by flow cytometry, and the correlation between NK cells and decompensation of liver function was analyzed. The frequency of circulating CD56bright NK cells was significantly increased while circulating CD56dim NK cells was significantly decreased in HBV-DLC patients as compared with HCs and HBV-CLC patients. Peripheral activated-CD56bright NK cells from HBV-DLC patients might express lower levels of inhibitory receptor CD158b1/2 and higher levels of activating receptor NKG2D and their expression of perforin and granzyme A/B also increased significantly compared with HCs, suggesting a high immune activation status of peripheral CD56bright NK cells in HBV-DLC patients. In HBV-DLC patients, the expression of CD107a and perforin in circulating CD56dim NK cells was positively correlated with cytolytic capacity while CD107a and perforin expression in circulating CD56dim NK cells were significantly decreased, suggesting an impaired cytolytic capacity of circulating CD56dim NK cells. Besides, we found that the perforin expression of circulating CD56dim NK cells correlated negatively with child-pugh classification in HBV-DLC patients. The functional properties of circulating NK cell subsets in HBV-DLC patients have changed significantly, especially of CD56dim NK cells which closely related to decompensation of liver function. These findings may help provide new perspectives and theoretical basis for the treatment of patients with HBV-DLC.

The transcription factor c-MYC (MYC thereafter) controls diverse transcription programs and plays a key role in the development of many human cancers. Cells develop multiple mechanisms to ensure that MYC levels and activity are precisely controlled in normal physiological context. As a short half-lived protein, MYC protein levels are tightly regulated by the ubiquitin proteasome system. Over a dozen of ubiquitin ligases have been found to ubiquitinate MYC whereas a number of deubiquitinating enzymes counteract this process. Recent studies show that SUMOylation and deSUMOylation can also regulate MYC protein stability and activity. Interestingly, evidence suggests an intriguing crosstalk between MYC ubiquitination and SUMOylation. Deregulation of the MYC ubiquitination-SUMOylation regulatory network may contribute to tumorigenesis. This review is intended to provide the current understanding of the complex regulation of the MYC biology by dynamic ubiquitination and SUMOylation and their crosstalk. Keywords: deSUMOylating enzymes, Deubiquitinating enzymes, MYC, SUMO, SUMOylation, Ubiquitination

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.

SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is affecting the whole world and threatening human health. We aim to investigate the immunological characteristics of monocytes in critical patients with COVID-19. METHODS: The number and immune status of monocytes were detected by flow cytometry in 32 COVID-19 patients and 18 healthy individuals. RESULTS: In critical patients with COVID-19, the absolute number of total monocytes and CD16- monocytes was significantly decreased but CD16+ pro-inflammatory monocytes was increased compared to healthy controls. Antigen presentation potential of monocytes, as measured by HLA-DR expression, was suppressed, while their inflammatory phenotype (CD38 expression) was enhanced. Cytokine levels showed sustained increases in critical patients. And the levels of IL-6 were positively correlated with CD16+ monocytes number. IL-6 and IL-10 levels were negatively correlated with HLA-DR expression of monocytes. During the recovery of COVID-19 patients, the count and immune status of monocyte subsets were restored by degrees. HLA-DR+ monocytes possessed good sensitivity and specificity for predicting the incidence of critical patients with COVID-19. CONCLUSIONS: In critical patients with COVID-19, decline in number and HLA-DR expression of monocytes might lead to decreased antigen presentation potential and thus immunosuppression, while increased CD16+ pro-inflammatory monocytes might mediate hyperinflammation. HLA-DR+ monocytes might be a meaningful assisted indicator to predict the incidence of critical patients with COVID-19.

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.

The stability and activity of the p53 tumor suppressor protein are tightly regulated by various posttranslational modifications, including SUMOylation. p53 can be modified by both SUMO1 and SUMO2, although how SUMOylation regulates p53 activity is still obscure. Whether p53 activity is directly regulated by deSUMOylation is also unclear. Here, we show that SENP1, a SUMO-specific protease implicated in pro-oncogenic roles, is a p53 deSUMOylating enzyme. SENP1 interacts with p53 and deSUMOylates p53 in cells and in vitro. Knockdown of SENP1 markedly induced p53 transactivation activity. We further show that SENP1 depletion synergizes with DNA damage-inducing agent etoposide to induce p53 activation and the expression of p21, leading to synergistic growth inhibition of cancer cells. Our results reveal that SENP1 is a critical p53 deSUMOylating enzyme and a promising therapeutic target in wild-type p53 containing cancer cells.

Chronic hepatitis B-related liver cirrhosis(HBV-LC)is the most common cirrhosis in China, which is characterized as liver damage and high mortality. We aim to investigate the characteristics of TRAILThirty cases each of chronic hepatitis B (CHB), HBV-related compensated liver cirrhosis (HBV-CLC) and HBV-related decompensated liver cirrhosis (HBV-DLC) patients were recruited in this study. Thirty age-and sex-matched healthy individuals were recruited as healthy controls (HCs). NK cell phenotypes were determined using flow cytometry. Serum chemokine concentrations were ascertained using the CBA Flex set. Cell apoptosis was analyzed using the Annexin V-PE/7-AAD apoptosis Kit.CD56The increase of CXCR3-related chemokines (including CXCL9, CXCL10 and CXCL11) might be related to the migration of TRAIL

Background Coronavirus disease 2019 (COVID-19) is posing a huge threat to human health worldwide. We aim to investigate the immune status of CD8+ T and NK cells in COVID-19 patients. Methods The count and immune status of lymphocytes were detected by flow cytometry in 32 COVID-19 patients and 18 healthy individuals. Results As the disease progression in COVID-19 patients, CD8+ T and NK cells were significantly decreased in absolute number but highly activated. After patients' condition improved, the count and immune status of CD8+ T and NK cells restored to some extent. GrA+CD8+ T and perforin+ NK cells had good sensitivity and specificity for assisting diagnosis of COVID-19. Conclusions As the disease progression, the declined lymphocytes in COVID-19 patients might lead to compensatory activation of CD8+ T and NK cells. GrA+CD8+ T and perforin+ NK cells might be used as meaningful indicators for assisting diagnosis of COVID-19., Highlights • As disease progression, CD8+ T and NK cells were decreased but highly activated. • After effective treatment, the immune status of CD8+ T and NK cells restored. • GrA+ CD8+ T and perforin+ NK cells could assist diagnosis of COVID-19.

NK cells play an important role in immune regulation and defense of infection, but their characteristics in patients with decompensated cirrhosis and their relationship with liver function remain unclear. We studied the functional properties of NK cells (including CD56

// Yingxiao Chen 1 , Yue-Gang Wang 1 , Yuhuang Li 1 , Xiao-Xin Sun 1 , Mu-Shui Dai 1 1 Department of Molecular and Medical Genetics, School of Medicine, and The OHSU Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97239, USA Correspondence to: Mu-Shui Dai, email: daim@ohsu.edu Keywords: deubiquitinating enzymes, MDMX, Otub1, p53, apoptosis Received: November 04, 2016 Accepted: December 20, 2016 Published: December 27, 2016 ABSTRACT Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2). However, whether Otub1 regulates MDMX stability and activity is not clear. Here we report that Otub1 also suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro , independently of its deubiquitinating enzyme activity. Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria-mediated apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53S46P, which was shown to be critical for p53’s mitochondrial function and apoptotic activity. Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion. Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria-mediated apoptosis, providing an alternative mechanism of Otub1’s role in apoptosis.

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

Highlights • The immune status of COVID-19 patients is different in each stage. • DN and DP cells are negatively correlated with IL-10 and IL-6, respectively. • Immune indexes help to distinguish COVID-19 and its severity early. • Dynamic immune monitoring can provide a reference for clinical drug selection., Background COVID-19 is threating human health worldwide. We aim to investigate the dynamic changes of immune status in COVID-19 patients with clinical evolution. Methods Sixty-one COVID-19 patients (42 mild cases and 19 severe cases, 51 cases without secondary infection as non-infection group and 10 cases with secondary bacterial/fungal infection as infection group) and 52 healthy controls (HCs) were enrolled from our hospital. Leucocyte classification, lymphocyte subsets and cytokines were detected by full-automatic blood cell analyzer and flow cytometer, respectively. Results Upon admission, eosinophils and lymphocyte subsets decreased significantly, while neutrophils, monocytes, basophils, IL-2, IL-6, IL-10 and IFN-γ increased significantly in COVID-19 patients compared to HCs. CD3+ T and DN (CD3+CD4-CD8-) cells appeared sustained decline, leucocytes, neutrophils and IL-10 showed sustained increase in severe group compared to mild group. Compared with the non-infection group, we observed a depletion of eosinophils, CD3+ T and CD4+ T cells, but leucocytes, neutrophils, IL-6 and IL-10 on the contrary in the infection group. Besides, in severe group of COVID-19 patients, DN cells were negatively correlated with IL-10, and DP (CD3+CD4+CD8+) cells were negatively correlated with IL-6. Lymphocytes, eosinophils, CD3+ T cells, CD4+ T cells, IL-6 and IL-10 all had great diagnostic efficacy (AUC, 0.905-0.975) for COVID-19. The laboratory indicators of COVID-19 patients with improved condition also showed a recovery trend with time. Conclusions The immune status of COVID-19 patients is different in each stage, and dynamic monitoring of related indicators can help predict the disease and may avoid cytokine storms.

Background Coronavirus disease 2019 (COVID-19) is posing a huge threat to human health worldwide. We aim to investigate the immune status of CD8+ T and NK cells in COVID-19 patients. Methods The count and immune status of lymphocytes were detected by flow cytometry in 32 COVID-19 patients and 18 healthy individuals. Results As the disease progression in COVID-19 patients, CD8+ T and NK cells were significantly decreased in absolute number but highly activated. After patients' condition improved, the count and immune status of CD8+ T and NK cells restored to some extent. GrA+CD8+ T and perforin+ NK cells had good sensitivity and specificity for assisting diagnosis of COVID-19. Conclusions As the disease progression, the declined lymphocytes in COVID-19 patients might lead to compensatory activation of CD8+ T and NK cells. GrA+CD8+ T and perforin+ NK cells might be used as meaningful indicators for assisting diagnosis of COVID-19.

Invariant NKT (iNKT) cells express markers of both T and NK cells and may produce various cytokines to regulate liver immunity. However, the role of iNKT cells in the progression of HBV-relative liver cirrhosis (HBV-LC) is incompletely understood. Here, we investigated the impact of peripheral iNKT cells on a cohort of patients with HBV-LC. The frequency, number, activation status, apoptosis and proliferation ability of peripheral iNKT cells were detected with flow cytometry. The impact of peripheral iNKT cells on the proliferation of hepatocyte cell line (MIHA) and activation of hepatic stellate cell line (LX-2) was detected with flow cytometry and PCR. In HBV-LC patients, the frequency and absolute number of peripheral iNKT cells significantly reduced, but the expression levels of CD25, interleukin (IL)-4, IL-13 and interferon (IFN)-γ increased. No difference was observed in the proliferation and apoptosis of circulating iNKT cells between patients and healthy controls (HCs). CXCR6 (CD186), known to be closely associated with iNKT cells migration from the periphery to the liver, was highly expressed on peripheral iNKT cells in HBV-LC patients. Furthermore, peripheral iNKT cells had a profound impact on MIHA cell proliferation and LX-2 cell activation through IL-4 or IL-13. Our data suggest that in HBV-LC patients, highly activated peripheral iNKT cells may migrate to the liver and affect hepatocyte cell line (MIHA) proliferation and hepatic stellate cell line (LX-2) activation through the expression of type 2 cytokines, which may result in excessive healing and contributing to the progression of fibrosis toward cirrhosis in liver.

Despite recent advances, the efficacy of androgen/androgen receptor (AR)-targeted therapy remains limited for many patients with metastatic prostate cancer. This is in part because prostate cancers adaptively switch to the androgen/AR-independent pathway for survival and growth, thereby conferring therapy resistance. Tumor hypoxia is considered as a major cause of treatment resistance. However, the exact mechanism is largely unclear. Here we report that chronic-androgen deprivation therapy (ADT) in the condition of hypoxia induces adaptive androgen/AR-independence, and therefore confers resistance to androgen/AR-targeted therapy, e.g., enzalutamide. Mechanistically, this is mediated by glucose-6-phosphate isomerase (GPI), which is transcriptionally repressed by AR in hypoxia, but restored and increased by AR inhibition. In turn, GPI maintains glucose metabolism and energy homeostasis in hypoxia by redirecting the glucose flux from androgen/AR-dependent pentose phosphate pathway (PPP) to hypoxia-induced glycolysis pathway, thereby reducing the growth inhibitory effect of enzalutamide. Inhibiting GPI overcomes the therapy resistance in hypoxia in vitro and increases enzalutamide efficacy in vivo., Prostate cancer often develops resistance to androgen receptor (AR) targeting drugs. Here, the authors show that, under conditions of hypoxia, AR inhibition via enzalutamide increases the expression of the glycolytic enzyme phosphoglucose isomerase (GPI) promoting a metabolic rewiring that allows the cells to survive, and consistent GPI inhibition restores sensitivity to enzalutamide.

Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca(2+) binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.

SUMOylation plays important roles in diverse cellular processes by regulating protein-protein interaction, protein localization, trafficking, stability and activity. p53 can by SUMOyalted at lysine (K) 384. Yet, how SUMOylation regulates p53 activity remain unclear. SUMOylation can be revered by a group of proteins called deSUMOylating enyzme including the SUMO specific protease (SENP) family proteins. How p53 is regulated by deSUMOylation is also not well understood. Here, we show that SENP1 directly interacts with and desumoylates p53 in cells and in vitro. Depletion of SENP1 by siRNA-mediated knockdown drastically increased the p53 transactivation activity without inducing p53 protein levels, resulting p53-depndent cell cycle arrast. Further, SENP1 knockdown significantly promoted p53 transactivation activity in cells following DNA damage. Additionally, we found that SENP1 binds to and deSUMOylates PML and aborageted the enhancing effect of PML on p53 SUMOylation. Thus, SNEP1 is novel p53 desumoylating enzyme that regulates p53 activity but not its stability. Citation Format: Mushui Dai, Krishna Chauhan, Yingxiao Chen, Xiao-Xin Sun. SENP1 is a p53 deSUMOylating enzyme and its depletion induces p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3536.

The stretching of smooth muscle tissue modulates contraction through augmentation of Ca(2+) transients, but the mechanism underlying stretch-induced Ca(2+) transients is still unknown. We found that mechanical stretching and maintenance of mouse urinary bladder smooth muscle strips and single myocytes at 30% and 18% beyond the initial length, respectively, resulted in Ca(2+) oscillations. Experiments indicated that mechanical stretching remarkably increased the production of nitric oxide (NO) as well as the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) oscillations and contractility increases were completely abolished by the NO inhibitor L-NAME or eNOS (also known as NOS3) gene inactivation. Moreover, exposure of eNOS-knockout myocytes to exogenous NO donor induced Ca(2+) oscillations. The stretch-induced Ca(2+) oscillations were greatly inhibited by the selective inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor xestospongin C and partially inhibited by ryanodine. Moreover, the stretch-induced Ca(2+) oscillations were also suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, but not by the soluble guanylyl cyclase (sGC) inhibitor ODQ. These results suggest that stretching myocyte and maintenance at a certain length results in Ca(2+) oscillations that are NO dependent , and sGC and cGMP independent, and results from the activation of PI3K in smooth muscle.

Cell motility plays a fundamental role in cancer metastasis. But the intracellular signaling mechanism that regulates cell motility remains unclarified. We have studied the functional role of intracellular Ca2+ release channel IP3 receptors(IP3Rs) and their novel negatively regulatory protein endoplasmic reticulum protein 44 (ERp44) in A549 cell migration. In wound healing assay, 30 μM 2-APB was used to inhibit Ca2+ release through the IP3Rs, and significantly decreased cell migration rates; while 20 μM Ryanodine did show any effect on cell migration. Immunofluorescence assay showed that cell polarization, membrane protrusion and formation of stress fiber of 2-APB treated A549 cells were inhibited 2h after wound producing. Consistent with this finding, the time-lapse imaging showed A549 cell became less motile after 2-APB treatment. Over expression of ERp44 in A549 cells inhibited 10 μM eATP-induced calcium activates and cell polarization, consequently inhibited cell migration both in wound healing assay and the time-lapse imaging. However, over expression of ERp44 in rat vascular smooth muscle, in which IP3Rs were not required for cell migration, did not inhibit cell polarization and cell migration. These findings suggest that IP3Rs are required for A549 cell migration and over expression of ERp44 inhibits cell polarization and cell migration through negatively regulating IP3Rs.Key Words Cell migration; ERp44; IP3Rs

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.

BACKGROUND Cardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca(2+) currents has not been investigated in developmental cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS We investigated the role of ANP in regulating L-type Ca(2+) channel current (I(CaL)) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I(CaL) in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of I(CaL) by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of I(CaL) induced by ANP was not affected by either Nomega, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of I(CaL) by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested. CONCLUSION/SIGNIFICANCES: Taken together, these results indicate that ANP induced depression of action potentials and I(CaL) is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3', 5'-cyclic monophophate (cAMP)-cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.