Flowchart: Preparation: Doc2
Text Box:   Doc2A-Doc2B


Text Box:  Ca(2+)



Vesicular trafficatng

Text Box: Egfp 

Text Box:  Muc13-1



Biochem Biophys Res Commun. 2000 Sep 24;276(2):626-32.Click here to read  Links

Doc2gamma, a third isoform of double C2 protein, lacking calcium-dependent phospholipid binding activity.

     Fukuda M, Mikoshiba K.

Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. mfukuda@brain.riken.go.jp

The Doc2 (double C2) family consists of two isoforms (Doc2alpha and Doc2beta) characterized by an N-terminal Munc13-1 interacting domain (Mid) and two C2 domains that interact with Ca(2+) and phospholipid at the C-terminus. This Ca(2+)-binding property is thought to be important to the regulation of neurotransmitter release. In this paper, we report a third isoform of mouse Doc2, named Doc2gamma. Doc2gamma also contains a putative Mid domain and two C2 domains, and it is 45.6 and 43.2% identical to mouse Doc2alpha and Doc2beta, respectively, at the amino acid level. In contrast to the other Doc2 isoforms, the C2 domains of Doc2gamma impair Ca(2+)-dependent phospholipid binding activity. The highest expression of Doc2gamma mRNA was found in the heart, but occurs ubiquitously, the same as Doc2beta. These findings indicate that Doc2gamma may also function as an effector for Munc13-1 and that it may be involved in the regulation of vesicular trafficking. Copyright 2000 Academic Press.

PMID: 11027523 [PubMed - indexed for MEDLINE]

Cell Calcium. 2006 Jan;39(1):85-93. Epub 2005 Nov 21.Click here to read  Links

Calcium concentration threshold and translocation kinetics of EGFP-DOC2B expressed in cultured Aplysia neurons.

     Malkinson G, Spira ME.

Department of Neurobiology, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

The double C2 domain protein family (DOC2) is characterized by two calcium-binding domains (C2). Upon binding to calcium, the affinity of the protein to phospholipids is significantly increased, leading to translocation of the protein from the cytosol to the plasma membrane. These properties, and the binding domain of DOC2B to Munc13, suggested that DOC2B could play a role in augmentation and potentiation of synaptic release. Nevertheless, the level of the free intracellular calcium concentration ([Ca(2+)](i)) which triggers its translocation under in vivo conditions, is not known. Using cultured Aplysia neurons that express rat EGFP-DOC2B, we found that the [Ca(2+)](i) increment necessary to induce EGFP-DOC2B translocation is approximately 200 nM in the bulk of the cytoplasm. The rate of EGFP-DOC2B recruitment to the plasma membrane is slower than the [Ca(2+)](i) elevation rate, while the detachment of EGFP-DOC2B from it is faster than the calcium removal. The extent of EGFP-DOC2B translocation to the plasma membrane reflects local submembrane [Ca(2+)](i). Our observations are consistent with the view that DOC2B can participate in the regulation of neurotransmitter release. It should be noted that EGFP-DOC2B could be used as a tool to map sub-membrane calcium dynamics under physiological conditions.

PMID: 16305808 [PubMed - indexed for MEDLINE]

J Neurochem. 2006 May;97(3):818-33. Epub 2006 Mar 3.Click here to read  Links

DOC2A and DOC2B are sensors for neuronal activity with unique calcium-dependent and kinetic properties.

     Groffen AJ, Friedrich R, Brian EC, Ashery U, Verhage M.

Department of Functional Genomics, Center for Neurogenomics and Cognition Research, Vrije Universiteit (VU) and VU Medical Centre, Amsterdam, the Netherlands. sander@cncr.vu.nl

Elevation of the intracellular calcium concentration ([Ca2+]i) to levels below 1 microm alters synaptic transmission and induces short-term plasticity. To identify calcium sensors involved in this signalling, we investigated soluble C2 domain-containing proteins and found that both DOC2A and DOC2B are modulated by submicromolar calcium levels. Fluorescent-tagged DOC2A and DOC2B translocated to plasma membranes after [Ca2+]i elevation. DOC2B translocation preceded DOC2A translocation in cells co-expressing both isoforms. Half-maximal translocation occurred at 450 and 175 nm[Ca2+]i for DOC2A and DOC2B, respectively. This large difference in calcium sensitivity was accompanied by a modest kinetic difference (halftimes, respectively, 2.6 and 2.0 s). The calcium sensitivity of DOC2 isoforms can be explained by predicted topologies of their C2A domains. Consistently, neutralization of aspartates D218 and D220 in DOC2B changed its calcium affinity. In neurones, both DOC2 isoforms were reversibly recruited to the plasma membrane during trains of action potentials. Consistent with its higher calcium sensitivity, DOC2B translocated at lower depolarization frequencies. Styryl dye uptake experiments in hippocampal neurones suggest that the overexpression of mutated DOC2B alters the synaptic activity. We conclude that both DOC2A and DOC2B are regulated by neuronal activity, and hypothesize that their calcium-dependent translocation may regulate synaptic activity.

PMID: 16515538 [PubMed - indexed for MEDLINE]



























Chronic exposure to TNF-alpha increases airway mucus gene expression in vivo.

Busse PJ, Zhang TF, Srivastava K, Lin BP, Schofield B, Sealfon SC, Li XM.

Division of Clinical Immunology, Mount Sinai School of Medicine, New York, NY 10029, USA. paula.busse@mssm.edu

BACKGROUND: Hypersecretion of mucus plays an important role in the pathogenesis and severity of asthma. The primary proteins in mucus are mucin glycoproteins; MUC-5AC is the primary airway mucin gene. The calcium chloride-activated channel gene hCLCA1 (gob-5 in the mouse) has been suggested to increase MUC-5AC gene expression, and both are increased in asthmatic patients and murine models. TNF-alpha increases the expression of these genes in vitro but has not been investigated in vivo. OBJECTIVE: We sought to determine whether TNF-alpha increases gene expression of gob-5 and MUC-5AC and induces mucus cell metaplasia in vivo. METHODS: Naive BALB/c mice received 50 ng of recombinant murine TNF-alpha (rmTNF-alpha) intratracheally daily for 1, 2, or 3 weeks; another group received the same dose of intratracheal rmTNF-alpha daily for 3 weeks and then alternate-day treatment for 3 additional weeks (total of 6 weeks). AKR mice received 50 ng of rmTNF-alpha intratracheally for 3 or 6 weeks daily. Naive nontreated mice were used as control animals. Airway gene products for gob-5 and MUC-5AC were determined by means of real-time PCR. Lung tissue sections were stained with periodic acid-Schiff/Alcian blue to assess mucus cell metaplasia. RESULTS: rmTNF-alpha significantly increased gene expression of airway gob-5 and MUC-5AC after 2 weeks in the BALB/c mice. There was noticeable mucus staining in all mice treated for at least 3 weeks with TNF-alpha and in 80% of the mice receiving 2 weeks of treatment. After 3 weeks of treatment, the AKR mice also showed increased gob-5 expression. CONCLUSIONS: This study demonstrates for the first time that TNF-alpha alone in vivo is sufficient to increase airway mucus gene expression in 2 murine strains.

PMID: 16337454 [PubMed - indexed for MEDLINE]

Am J Respir Crit Care Med. 2005 Feb 15;171(4):305-14. Epub 2004 Nov 5.

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Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation.

Deshmukh HS, Case LM, Wesselkamper SC, Borchers MT, Martin LD, Shertzer HG, Nadel JA, Leikauf GD.

University of Cincinnati, P.O. Box 670056, Cincinnati, OH 45267-0056, USA.

Chronic obstructive pulmonary disease is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke, increases mucin 5AC (MUC5AC), a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses of 10 muM or greater, higher than those sufficient (0.03 muM) to increase MUC5AC mRNA, suggesting that MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small, interfering RNA directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase. Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloproteinase 3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Together, these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.

PMID: 15531749 [PubMed - indexed for MEDLINE]


Zhonghua Jie He He Hu Xi Za Zhi. 2004 Sep;27(9):585-8.

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[Expression of epidermal growth factor receptor and MUC5AC on human airway with chronic obstructive pulmonary disease]

[Article in Chinese]

Mao L, Bai CX, Zhang M, Wang YH, Chen J.

Department of Pulmonary Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

OBJECTIVE: To determine the relationship between epidermal growth factor receptor (EGFR) expression and mucin 5AC (MUC5AC) synthesis in human airways with chronic obstructive pulmonary disease (COPD). METHODS: Lung specimens were obtained from 38 patients who were undergoing lobectomy, and the samples were taken from areas remote to the lesion. EGFR and MUC5AC protein expression were examined using immunohistochemical analysis and Western blot in peripheral airways (less than 2 mm in diameter) from 16 subjects with COPD, 10 subjects with a history of more than 30 pack-year smoking and 12 nonsmokers or exsmokers (0 - 10 pack-year smoker). RESULTS: Weak EGFR protein signals were detected in the lungs of the controls (2.01 +/- 1.02) in comparison with stronger signal in the COPD patients (4.62 +/- 1.65, P < 0.01) and the smokers (4.89 +/- 1.89, P < 0.01). EGFR immunoreactivity was observed mainly in goblet cells in the controls, the percentage of positive cell being 71.1% +/- 14.3%, which was higher than those in COPD (21.1% +/- 8.6%) or in the smoker (21.9% +/- 9.7%) airways. In contrast, COPD or smoker airways showed more expression of EGFR which expressed mainly in basal cells (42.9% +/- 14.2%, 52.1% +/- 13.5%, respectively) than that in the control airways (23.7% +/- 9.5%, P < 0.01). However there was no significant differences in EGFR expression and location in peripheral airways between COPD patients and smokers (P > 0.05). There was significant positive correlation between EGFR immunoreactivity and the area of the MUC5AC positive staining in all subjects (r = 0.877 4, P < 0.001). CONCLUSION: It is suggested that EGFR activation is involved in mucin expression in COPD airways.

PMID: 15498267 [PubMed - indexed for MEDLINE]























Differential Regulation of CD40-Mediated TNF Receptor-Associated Factor Degradation in B Lymphocytes.

Moore CR, Bishop GA.

Interdisciplinary Graduate Program in Immunology.

Engagement of CD40 on murine B cells by its ligand CD154 induces the binding of TNFR-associated factors (TRAFs) 1, 2, 3, and 6, followed by the rapid degradation of TRAFs 2 and 3. TRAF degradation occurs in response to signaling by other TNFR superfamily members, and is likely to be a normal regulatory component of signaling by this receptor family. In this study, we found that receptor-induced TRAF degradation limits TRAF2-dependent CD40 signals to murine B cells. However, TRAFs 1 and 6 are not degraded in response to CD40 engagement, despite their association with CD40. To better understand the mechanisms underlying differential TRAF degradation, mixed protein domain TRAF chimeras were analyzed in murine B cells. Chimeras containing the TRAF2 zinc (Zn) domains induced effective degradation, if attached to a TRAF domain that binds to the PXQXT motif of CD40. However, the Zn domains of TRAF3 and TRAF6 could not induce degradation in response to CD40, regardless of the TRAF domains to which they were attached. Our data indicate that TRAF2 serves as the master regulator of TRAF degradation in response to CD40 signaling, and this function is dependent upon both the TRAF Zn domains and receptor binding position.

PMID: 16148124 [PubMed - in process]



B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells.

Yang M, Hase H, Legarda-Addison D, Varughese L, Seed B, Ting AT.

Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029.

B cell maturation Ag (BCMA), a member of the TNFR superfamily expressed on B cells, binds to a proliferation-inducing ligand (APRIL) and B cell-activating factor of the TNF family (BAFF) but the specific B cell responses regulated by BCMA remain unclear. This study demonstrates that ligation of A20 B cells transfected with BCMA induces the expression of CD40, CD80/B7-1, CD86/B7-2, MHC class II, and CD54/ICAM-1, which subsequently enhances the presentation of OVA peptide Ag to DO11.10 T cells. BCMA expression in murine splenic B cells can be induced with IL-4 and IL-6, allowing subsequent treatment with APRIL or agonist anti-BCMA to similarly induce Ag presentation. A comparative analysis of hybrid receptors of TNFR2 fused to the cytoplasmic domains of APRIL/BAFF receptors found that only BCMA, but not transmembrane activator and calcium-modulator and cyclophilin ligand interactor or BAFF-R, is capable of activating Ag presentation. Although all three receptors can trigger NF-kappaB signaling, only BCMA activates the JNK pathway conferring on BCMA the specific ability to activate this Ag presentation response.

PMID: 16116167 [PubMed - in process]






ICAM-1 gene expression in endothelial cells: Effects on the inhibition of STAT3 phosphorylation.
Resveratrol suppresses IL-6-induced
Wung BS, Hsu MC, Wu CC, Hsieh CW.

Department of Applied Microbiology, National Chiayi University, Chiayi, Taiwan.

Resveratrol, a polyphenolic phytoaxelin present in red wine, has been suggested to protect against atherosclerosis and cardiovascular disease because of its antioxidant effects. Intercellular adhesion molecule (ICAM-1), induced by cytokines, has been hypothesized to play a role in the early events during atherosclerosis. In this study we tested the effects of resveratrol upon both IL-6-induced ICAM-1 gene expression and its underlying signaling pathways in endothelial cells (ECs). Resveratrol was found to inhibit both TNFalpha- and IL-6-induced ICAM-1 gene expression at the promoter, transcriptional and protein levels. Resveratrol also abrogates the tyr705 phosphorylation of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner. Although quercetin had similar effects, resveratrol showed higher inhibitory properties following 2-4 h pretreatments. Resveratrol has been shown to induce the activity of endothelial nitric oxide synthase (eNOS) and increase NO production. Consistent with this, the treatment of ECs with a NO donor (SNAP) reduces IL-6-induced STAT3 phosphorylation. Conversely, exposure of ECs to a NOS inhibitor reversed the effects of resveratrol upon IL-6-induced STAT3 phosphorylation. Furthermore, ECs transfected with constitutively active Rac1 (RacV12) showed increases in ICAM-1 promoter activity, intracellular reactive oxygen species (ROS) levels and STAT3 phosphorylation, and these increases were attenuated by resveratrol treatment. In summary, we demonstrate for the first time that resveratrol inhibits IL-6-induced ICAM-1 gene expression, in part, by interfering with Rac-mediated pathways via the attenuation of STAT3 phosphorylation. This study therefore provides important new insights that may contribute to the proposed beneficial effects of resveratrol in endothelial responses to cytokines during inflammation.

PMID: 16150460 [PubMed - as supplied by publisher]