| 1. |
- Bankell, Elisabeth, et al.
(författare)
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LL-37-induced caspase-independent apoptosis is associated with plasma membrane permeabilization in human osteoblast-like cells
- 2021
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Ingår i: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 135
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Tidskriftsartikel (refereegranskat)abstract
- The host defense peptide LL-37 is active against both gram-positive and gram-negative bacteria, but it has also been shown to reduce human host cell viability. However, the mechanisms behind LL-37-induced human host cell cytotoxicity are not yet fully understood. Here, we assess if LL-37-evoked attenuation of human osteoblast-like MG63 cell viability is associated with apoptosis, and if the underlying mechanism may involve LL-37-induced plasma membrane permeabilization. MG63 cell viability and plasma membrane permeabilization were investigated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and by measuring lactate dehydrogenase (LDH) release, respectively. Apoptosis was assessed by the terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) assay and Annexin V flow cytometry, and caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage were determined by Western blot. LL-37 (4 and 10 μM) reduced both cell number and cell viability, and these effects were associated with a pro-apoptotic effect demonstrated by positive TUNEL staining and Annexin V flow cytometry. LL-37-induced apoptosis was not coupled to either caspase-3 or PARP cleavage, suggesting that LL-37 causes caspase-independent apoptosis in MG63 cells. Both LL-37 and the well-known plasma membrane permeabilizer Triton X-100 reduced cell viability and stimulated LDH release. Triton X-100-treated cells showed positive TUNEL staining, and the detergent accumulated cells in late apoptosis/necrosis. Similar to LL-37, Triton X-100 caused no PARP cleavage. We conclude that LL-37 promotes caspase-independent apoptosis, and that this effect seems coupled to plasma membrane permeabilization in human MG63 cells.
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| 2. |
- Dahl, Sara, et al.
(författare)
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The host defense peptide LL-37 triggers release of nucleic acids from human mast cells
- 2018
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Ingår i: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 109, s. 39-45
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Tidskriftsartikel (refereegranskat)abstract
- The human host defense peptide LL-37 possesses antimicrobial activity but also affects host cell function and viability. Mast cells are involved in innate immunity but no data have been presented on effects of LL-37 on human mast cell viability and export of nucleic acids. Here, we demonstrated by immunofluorescence microscopy that synthesized LL-37 was internalized by human LAD2 mast cells and detected both in cytoplasm and nucleus. Treatment with high (4 and 10 μM) but not low (1 μM) concentrations of LL-37 for 4 h reduced cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Stimulation with 10 μM LL-37 for 4 h enhanced export of nucleic acids, total protein and lactate dehydrogenase (LDH), suggesting that both nuclear and plasma membranes are permeabilized by LL-37. Although LL-37 triggered release of nucleic acids, no extracellular trap-like structures were observed by laser scanning confocal microscopy of cells incubated with the plasma membrane impermeable nucleic acid fluorophore SYTOX-Green, indicating that LL-37 promotes export of nucleic acids but not formation of extracellular traps. On the other hand, phorbol-12-myristate-13-acetate (PMA), which is a well-known inducer of extracellular traps, stimulated export of nucleic acids and also formation of extracellular trap-like structures. However, PMA had no effect on export of either total protein or LDH. Hence, LL-37 and PMA seem to stimulate export of nucleic acids from LAD2 mast cells through different pathways. In conclusion, we demonstrate that LL-37 triggers release of nucleic acids from human mast cells but not the formation of extracellular trap-like structures.
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| 3. |
- Svensson, Daniel, et al.
(författare)
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Inhibition of MicroRNA-125a Promotes Human Endothelial Cell Proliferation and Viability through an Antiapoptotic Mechanism.
- 2014
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Ingår i: Journal of Vascular Research. - : S. Karger AG. - 1423-0135 .- 1018-1172. ; 51:3, s. 239-245
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Tidskriftsartikel (refereegranskat)abstract
- The microRNA-125a (miR-125a) is highly expressed in endothelial cells, but its role in vascular biology is not known. Endothelial cell proliferation and viability play an important role in endothelial healing, and we hypothesize that miR-125a regulates this process. The aim of the present study was to investigate if miR-125a controls human endothelial cell proliferation, viability and endothelial healing, and to assess the mechanisms involved. We showed that overexpression of miR-125a by transfection with miR-125a mimic reduced human umbilical vein endothelial cell (HUVEC) proliferation and viability, and stimulated apoptosis as demonstrated by a miR-125a-induced increase of the proportion of annexin V-positive cells monitored by flow cytometry. Moreover, we showed that the miR-125a mimic downregulated the antiapoptotic Bcl2 protein and upregulated caspase 3, suggesting that these two proteins represent molecular targets for miR-125a. Accordingly, transfection with miR-125a inhibitor, downregulating miR-125a expression, promoted HUVEC proliferation and viability, and reduced apoptosis. Importantly, transfection with miR-125a inhibitor promoted HUVEC tube formation in Matrigel, suggesting that reduction of miR-125a has a proangiogenic effect. In conclusion, downregulation of miR-125a through local transfection with miR-125a inhibitor might be a new way to enhance endothelial cell proliferation and viability, thereby promoting the reendothelialization observed in response to intimal injury. © 2014 S. Karger AG, Basel.
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| 4. |
- Säll, Johanna, et al.
(författare)
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The antimicrobial peptide LL-37 alters human osteoblast Ca2+ handling and induces Ca2+-independent apoptosis
- 2013
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Ingår i: Journal of Innate Immunity. - : S. Karger. - 1662-811X .- 1662-8128. ; 3:5, s. 290-300
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Tidskriftsartikel (refereegranskat)abstract
- The human antimicrobial peptide cathelicidin LL-37 has, besides its antimicrobial properties, also been shown to regulate apoptosis in a cell type-specific manner. Mechanisms involved in LL-37-regulated apoptotic signaling are not identified. Here, we show that LL-37 reduces the human osteoblast-like MG63 cell number and cell viability in the micromolar concentration range with an IC50 value of about 5 µM. Treatment with 4 µM LL-37 increased the number of annexin V-positive cells and stimulated activation of caspase 3 showing that LL-37 promotes apoptosis. Treatment with 4 µM LL-37 caused an acute and sustained rise in intracellular Ca(2+) concentration assessed by laser-scanning confocal microscopy of Fluo-4-AM-loaded MG63 cells. LL-37 increased Ca(2+) also in the presence of the respective L- and T-type voltage-sensitive Ca(2+) channel blockers nifedipine and NiCl2. LL-37 had no effect on Ca(2+) in cells incubated with Ca(2+)-free solution. LL-37 (4 and 8 µM) reduced the MG63 cell number both in the presence and absence of Ca(2+) in the medium. In conclusion, LL-37 reduces the osteoblast cell number by promoting apoptosis, and furthermore, LL-37 stimulates Ca(2+) inflow via a mechanism independent of voltage-sensitive Ca(2+) channels. Interestingly, LL-37-induced lowering of the cell number seems to be mediated via a mechanism independent of Ca(2+).
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| 5. |
- Aidoukovitch, Alexandra, et al.
(författare)
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Antimicrobial peptide LL-37 and its pro-form, hCAP18, in desquamated epithelial cells of human whole saliva
- 2020
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Ingår i: European Journal of Oral Sciences. - : Wiley. - 0909-8836 .- 1600-0722. ; 128:1, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- The antimicrobial peptide LL-37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL-37 is formed from its intracellular pro-form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell-containing and cell-free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL-37 in these fractions using ELISA. Cellular expression of hCAP18/LL-37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell-free saliva contained hCAP18/LL-37. Western blot analysis of cell-pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL-37. Phase-contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL-37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL-37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL-37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.
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| 6. |
- Aidoukovitch, Alexandra, et al.
(författare)
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Strontium chloride promotes cell proliferation in a human osteoblast cell line
- 2014
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Strontium ranelate (SrRan) is the active component of drugs currently used for reducing the risk of fractures in patients suffering from osteoporosis. Despite extensive use, the underlying mechanisms of action of Sr2+ are not fully understood. In the present study, we assess the impact of SrCl2 on human osteoblast activity and proliferation. Cultures of the human osteoblast-like cell line MG63 were treated for 72 h in presence of 0.1 mM, 1 mM, 5 mM and 10 mM SrCl2 or vehicle, used in control groups. Cells were counted manually using a Bürker chamber. Total protein content was determined by colorimetric analysis performed by a microplate reader using Bio-Rad protein assay. Alkaline phosphatase (ALP) activity was determined enzymatically and normalized to total protein content in each sample. Cell viability was assessed using the MTT assay. Treatment with 5 mM SrCl2 for 72 h enhanced total MG63 cell protein content by 37% compared to controls (p<0.01). A lower concentration (0.1 mM) of SrCl2 had no effect on total protein. Incubation with 5 mM SrCl2 for 72 h increased MG63 cell number by 38% compared to controls (p<0.001). The SrCl2-induced increase in cell number was associated with enhanced (+14% compared to controls, p<0.05) cell viability. Treatment with a higher concentration (10 mM) of SrCl2 enhanced cell number similar to 5 mM SrCl2 (+54% compared to controls, p<0.05). Treatment with 0.1 or 5 mM SrCl2 for 72 h had no effect (p>0.05) on MG63 cell ALP activity, while 1 mM SrCl2 reduced ALP activity as well as total protein content by about 25% compared to controls (p<0.05). The current results demonstrate that treatment with SrCl2 for 72 h, at concentrations higher than 1 mM promotes cell proliferation in human osteoblast-like cells, suggesting that Sr2+ may enhance bone formation through this mechanism.
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| 7. |
- Aidoukovitch, Alexandra, et al.
(författare)
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The host defense peptide LL-37 is internalized by human periodontal ligament cells and prevents LPS-induced MCP-1 production
- 2019
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Ingår i: Journal of Periodontal Research. - : Wiley. - 0022-3484 .- 1600-0765. ; 54:6, s. 662-670
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Tidskriftsartikel (refereegranskat)abstract
- Objective: The human host defense peptide LL-37 both shows antimicrobial effects and modulates host cell properties. Here, we assess the effects of synthesized LL-37 on lipopolysaccharide (LPS)-induced inflammation in human periodontal ligament (PDL) cells and investigates underlying mechanisms. Background: LL-37 has been detected in the periodontal tissues, but its functional importance for PDL cell innate immune responses is not known. Methods: Human PDL cells were obtained from premolars extracted on orthodontic indications. Cellular pro-inflammatory monocyte chemoattractant protein-1 (MCP-1) mRNA expression was determined using quantitative real-time RT-PCR. MCP-1 protein production was assessed by western blot and ELISA. Internalization of LL-37 by PDL cells was visualized by immunocytochemistry. Nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activity was assessed by western blot of phosphorylated p65, phosphorylated p105, and IκBα proteins. Binding of LL-37 to PDL cell DNA was determined by isolation and purification of DNA and dot blot for LL-37 immunoreactivity. Results: Treatment with LL-37 (1 µmol/L) for 24 hours prevented LPS-induced stimulation of MCP-1 expression analyzed both on transcript and on protein levels. Stimulation with LL-37 (1 µmol/L) for 24 hours had no effect on toll-like receptor (TLR)2 and TLR4 transcript expression, suggesting that LL-37 acts downstream of the TLRs. Preincubation with LL-37 for 60 minutes followed by stimulation with LPS for 24 hours in the absence of LL-37 completely prevented LPS-evoked MCP-1 transcript expression, implying that LL-37 acts intracellularly and not via binding and neutralization of LPS. In PDL cells stimulated with LL-37 for 60 minutes, the peptide was internalized as demonstrated by immunocytochemistry, suggesting an intracellular mechanism of action. LL-37 immunoreactivity was observed both in the cytosol and in the nucleus. Downregulation of LPS-induced MCP-1 by LL-37 was not mediated by reduction in NF-κB activity as shown by unaltered expression of phosphorylated p65, phosphorylated p105, and IκBα NF-κB proteins in the presence of LL-37. Immunoreactivity for LL-37 was observed in PDL cell DNA treated with but not without 0.1 and 1 µmol/L LL-37 for 60 minutes in vitro. Conclusion: LL-37 abolishes LPS-induced MCP-1 production in human PDL cells through an intracellular, NF-κB-independent mechanism which probably involves direct interaction between LL-37 and DNA.
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| 8. |
- Aidoukovitch, Alexandra, et al.
(författare)
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Vitamin D triggers hCAP18/LL-37 production : Implications for LL-37-induced human osteoblast cytotoxicity
- 2024
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Ingår i: Biochemical and Biophysical Research Communications. - 0006-291X. ; 712-713
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Tidskriftsartikel (refereegranskat)abstract
- The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3–4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 μM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.
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| 9. |
- Anders, Emma, et al.
(författare)
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Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1 : a novel mechanism for regulation of MCP-1 production and function
- 2018
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Ingår i: Biochemical Journal. - 0264-6021. ; 475:4, s. 775-786
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Tidskriftsartikel (refereegranskat)abstract
- The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/ p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.
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| 10. |
- Anders, Emma, et al.
(författare)
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LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis
- 2018
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 501:1, s. 280-285
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Tidskriftsartikel (refereegranskat)abstract
- The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.
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