• Molecules and Cells
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• v.44 no.10
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• pp.758-769
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• 2021

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I_CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I_CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I_CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I_CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.

• Molecules and Cells
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• v.26 no.1
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• pp.48-52
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• 2008

We here demonstrate an anti-inflammatory action of raloxifene, a selective estrogen receptor modulator, in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells. Treatment with raloxifene at micromolar concentrations suppressed the production of nitric oxide (NO) by down-regulating expression of the inducible nitric oxide synthase (iNOS) gene in LPS-activated cells. The decreased expression of iNOS and subsequent reduction of NO were due to inhibition of nuclear translocation of transcription factor NF-${\kappa}B$. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. In addition, pretreatment with raloxifene reduced LPS-induced Akt phosphorylation as well as NF-${\kappa}B$ DNA binding activity and NF-${\kappa}B$-dependent reporter gene activity. Thus our findings indicate that raloxifene exerts its anti-inflammatory action in LPS-stimulated macrophages by blocking the PI 3-kinase-Akt-NF-${\kappa}B$ signaling cascade, and eventually reduces expression of pro-inflammatory genes such as iNOS.

• IMMUNE NETWORK
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• v.13 no.1
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• pp.1-9
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• 2013

Autophagy is a fundamental cellular process in eukaryotic cells for maintaining homeostasis by degrading cellular proteins and organelles. Recently, the roles of autophagy have been expanded to immune systems, which in turn modulate innate immune responses. More specifically, autophagy acts as a direct effector for protection against pathogens, as well as a modulator of pathogen recognition and downstream signaling in innate immune responses. In addition, autophagy controls autoimmunity and inflammatory disorders by negative regulation of immune signaling. In this review, we focus on recent advances in the role of autophagy in innate immune systems.

• Molecules and Cells
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• v.27 no.1
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• pp.47-54
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• 2009

A cDNA clone for a transcript preferentially expressed during an early phase of flooding was isolated from Nicotiana tabacum. Nucleotide sequencing of the cDNA clone identified an open reading frame that has high homology to the previously reported glycine-rich RNA-binding proteins. The open reading frame consists of 157 amino acids with an N-terminal RNA-recognition motif and a C-terminal glycine-rich domain, and thus the cDNA clone was designated as Nicotiana tabaccum glycine-rich RNA-binding protein-1 (NtGRP1). Expression of NtGRP1 was upregulated under flooding stress and also increased, but at much lower levels, under conditions of cold, drought, heat, high salt content, and abscisic acid treatment. RNA homopolymer-binding assay showed that NtGRP1 binds to all the RNA homopolymers tested with a higher affinity to poly r(G) and poly r(A) than to poly r(U) and poly r(C). Nucleic acid-binding assays showed that NtGRP1 binds to ssDNA, dsDNA, and mRNA. NtGRP1 suppressed expression of the fire luciferase gene in vitro, and the suppression of luciferase gene expression could be rescued by addition of oligonucleotides. Collectively, the data suggest NtGRP1 as a negative modulator of gene expression by binding to DNA or RNA in bulk that could be advantageous for plants in a stress condition like flooding.

• Biomolecules & Therapeutics
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• v.6 no.3
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• pp.276-282
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• 1998

Retinoids regulate a wide variety of biological processes such as cellular proliferation and differentiation in many cell types. They have also shown to stimulate replication of several viruses including human cytomegalovirus (CMV). Retinoid signalling pathway involves two distinct subfamilies of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that bind to specific retinoic acid response elements (RAREs) in the promoter regions of retinoid-target genes. Here, we characterized RAREs in the regulatory regions of the CMV and of the hepatitis B vi.us (HBV). The viral RAREs, i.e., CMV-RARE and HBV-RARE, are composed of two consensus RARE half-sites (A/GGGTCA) arranged as a direct repeat separated by 5-bp and 1-bp, respectively. The RAREs were activated by both RAR/RXR heterodimers and RXR homodimers in transient transfection experiments. We also found that COUP-TF$\alpha$ (chicken ovalbumin upstream promoter-transcription factor u) and COUP-TF$\beta$ repressed the retinoid response of the viral elements. Further we demonstrated that previously known retinoid antagonist, SRI 1330, repressed retinoid-induced transactivation of the CMV-RARE. These results implicate Vitamin A, it's nuclear receptors and COUP-TFs as important regulators of the CMV and HBV pathogenesis and the SRl1330 as potential negative modulator of such retinoid-dependent processes.

• Journal of Ginseng Research
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• v.43 no.4
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• pp.527-538
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• 2019

Background: Ginsenoside Rg1 was shown to exert ligand-independent activation of estrogen receptor (ER) via mitogen-activated protein kinase-mediated pathway. Our study aimed to delineate the mechanisms by which Rg1 activates the rapid ER signaling pathways. Methods: ER-positive human breast cancer MCF-7 cells and ER-negative human embryonic kidney HEK293 cells were treated with Rg1 ($10^{-12}M$, $10^{-8}M$), $17{\beta}$-estradiol ($10^{-8}M$), or vehicle. Immunoprecipitation was conducted to investigate the interactions between signaling protein and ER in MCF-7 cells. To determine the roles of these signaling proteins in the actions of Rg1, small interfering RNA or their inhibitors were applied. Results: Rg1 rapidly induced $ER{\alpha}$ translocation to plasma membrane via caveolin-1 and the formation of signaling complex involving linker protein (Shc), insulin-like growth factor-I receptor, modulator of nongenomic activity of ER (MNAR), $ER{\alpha}$, and cellular nonreceptor tyrosine kinase (c-Src) in MCF-7 cells. The induction of extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase (MEK) phosphorylation in MCF-7 cells by Rg1 was suppressed by cotreatment with small interfering RNA against these signaling proteins. The stimulatory effects of Rg1 on MEK phosphorylation in these cells were suppressed by both PP2 (Src kinase inhibitor) and AG1478 [epidermal growth factor receptor (EGFR) inhibitor]. In addition, Rg1-induced estrogenic activities, EGFR and MEK phosphorylation in MCF-7 cells were abolished by cotreatment with G15 (G protein-coupled estrogen receptor-1 antagonist). The increase in intracellular cyclic AMP accumulation, but not Ca mobilization, in MCF-7 cells by Rg1 could be abolished by G15. Conclusion: Ginsenoside Rg1 exerted estrogenic actions by rapidly inducing the formation of ER containing signalosome in MCF-7 cells. Additionally, Rg1 could activate EGFR and c-Src ER-independently and exert estrogenic effects via rapid activation of membrane-associated ER and G protein-coupled estrogen receptor.