scholarly journals Oxidized low-density lipoprotein enhances Orai3 expression levels to increase proliferation of human lens epithelial cells

2020 ◽  
Author(s):  
Ru Zhang ◽  
Suwen Bai ◽  
Yang Fang ◽  
Zhixin Zhou ◽  
Linghui Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Low Density ◽  
Oxidized Low Density Lipoprotein ◽  
Expression Levels ◽  
Human Lens Epithelial Cells

Abstract Background Serum lipid levels, especially of oxidized low-density lipoprotein (oxLDL), are related to the development of cataracts, but the mechanisms underlying the role of oxLDL in cataract development in human lens epithelial cells (HLEpiCs) remain unclear. Calcium (Ca2+) overload is also known to be involved in lens turbidity. Store-operated Ca2+ entry (SOCE) is an important pathway mediating Ca2+ influx in lens epithelial cells. The Orai family proteins, which form a type of SOCE channel, localize at the plasma membrane and control Ca2+ influx in response to the depletion of Ca2+ stores in the endoplasmic reticulum. Methods In the present study, we used RNA sequencing, western blot analyses, cell proliferation assays, Ca2+ measurements, and small interfering (si)RNA transfections to investigate the effects of oxLDL on SOCE and proliferation of human lens epithelial cells (HLEpiCs). Results The key findings of our study that suggest this conclusion are that (1) oxLDL enhances the expression levels of Orai3, but not Orai1 or Orai2, in HLEpiCs in vitro; (2) oxLDL significantly increases the proliferation of HLEpiCs in a concentration-dependent manner; (3) oxLDL significantly increases ATP-induced SOCE without affecting Ca2+ release in HLEpiCs; and (4) knockdown of Orai3 significantly reduces cell proliferation and ATP-induced SOCE in HLEpiCs. Conclusions These findings suggest that Ca2+ signaling altered by overexpression of Orai3 may be a mechanism whereby oxLDL increases HLEpiC proliferation to contribute to the development of cataract. Thus, Orai3 may be a target warranting development for the treatment of cataract associated with obesity or hyperlipidemia.

scholarly journals The effects of c-Src kinase on EMT signaling pathway in human lens epithelial cells associated with lens diseases

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xingyu Li ◽  
Fang Wang ◽  
Meixia Ren ◽  
Minjuan Du ◽  
Jian Zhou
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Western Blot ◽  
Src Kinase ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Protein Levels ◽  
Human Lens Epithelial Cells ◽  
And Migration ◽  
E Cadherin

Abstract Background The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors. Methods Human lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-SrcY530F to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA, and Vimentin were examined at 48 h by RT-PCR and western blot. At 48 h and 72 h of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assays. Results Activity of c-Src kinase, which causes the expression of p-Src418, was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-SrcY530F, the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src418 increased. The expressions of both E-cadherin and ZO-1 were suppressed, while the expressions of vimentin and α-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src418 expressions were knocked down. The expressions of E-cadherin and ZO-1 increased, but the expressions of Vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced. Conclusions The c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.

scholarly journals Lectin-like, oxidized low-density lipoprotein receptor-1-deficient mice show resistance to age-related knee osteoarthritis

2017 ◽  
Author(s):  
Kazuhiko Hashimoto ◽  
Yutaka Oda ◽  
Fumihisa Nakamura ◽  
Ryosuke Kakinoki ◽  
Masao Akagi
Keyword(s):  
Articular Chondrocytes ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Oxidized Low Density Lipoprotein ◽  
Expression Levels ◽  
Knee Oa ◽  
Age Related ◽  
Significant Difference ◽  
The Mean

The lectin-like, oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1)/ox-LDL system contributes to atherosclerosis and may be involved in cartilage degeneration. The purpose of this study was to determine whether the LOX-1/ox-LDL system contributes to age-related osteoarthritis (OA) in vivo, using LOX-1 knockout (LOX-1 KO) mice. Knee cartilage from 6, 12, and 18-month old (n = 10/group) C57Bl/6 wild-type (WT) and LOX-1 KO mice was evaluated by determining the Osteoarthritis Research Society International (OARSI) score of Safranin-O stained samples. The prevalence of knee OA in both mouse strains was also investigated. Expression levels of LOX-1, ox-LDL, runt-related transcription factor-2 (Runx2), type-X collagen (COL X), and matrix metalloproteinase-13 (MMP-13) in the articular chondrocytes were analyzed immunohistologically. No significant difference was observed in the mean scores of WT (2.00±0.61) and LOX-1 KO mice (2.00±0.49) at 6 months of age (P=1.00, n=10). At 12 and 18 months of age, the mean scores of LOX-1 KO mice (3.75±0.93 and 5.50±0.78) were significantly lower than those of WT mice (5.25±1.14 and 9.00±1.01; P<0.001 in both cases; n=10). The prevalence of OA in LOX-1 KO mice was lower than that in WT mice at 12 and 18 months of age (40 vs 70%, 70 vs 90%, respectively; n=10). The expression levels of Runx2, COL X, and MMP-13 in articular chondrocytes significantly decreased in LOX-1 KO, mice compared with those in WT mice. The study indicated that the LOX-1/ox-LDL system in chondrocytes plays a role in the pathogenesis of age-related knee OA, which is potentially a target for preventing OA progression.

scholarly journals Short-Wavelength Blue Light Contributes to Pyroptosis of Human Lens Epithelial Cells (hLECs) by Caspase-1-GSDMD Signalling Axis Activation

2020 ◽  
Author(s):  
Zhaowei Song ◽  
Xiaohui Wang ◽  
Huazhang Li ◽  
Ying Sun ◽  
Kexin Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Epithelial Cells ◽  
Blue Light ◽  
Short Wavelength ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Expression Levels ◽  
Human Lens Epithelial Cells ◽  
Caspase 1

Abstract Backgroud: To examine the effects of short-wavelength blue light (SWBL) on cultured human lens epithelial cells (hLECs). The nosogenesis of cataracts after SWBL exposure was discussed. Methods: HLE-B3 hLECs were divided into 3 groups randomly: A: normal control group, which consisted of hLECs cultured in the dark; B: the caspase-1 inhibitor group; and C: the SWBL exposure group. After the SWBL (2500 lux) irradiation (for 8, 16, 24, and 32 h), the caspase-1 and gasdermin D (GSDMD) expression levels in HLE-B3 hLECs were examined using ELISA, immunofluorescence, and Western blotting analyses. Double-positive staining of HLE-B3 hLECs for activated and inhibited caspase-1 was used to confirm pyroptosis in hLECs by flow cytometry. Results: SWBL can cause cell death in HLE-B3 hLECs, but a caspase-1 inhibitor suppressed cell death. The flow cytometry results also confirmed the does-dependent of short-wavelength blue light irradiation on pyroptotic death of hLECs. Caspase-1 and GSDMD expression levels of all hLECs groups changed with short-wavelength blue light exposure times (8, 16, 24, and 32 h) and were higher in groups B and C than group A. The immunofluorescence results demonstrated that the expression of GSDMD-N was higher in the cell membrane in both the B and C groups than in the A group.Conclusion: The data indicate that SWBL induces pyroptotic programmed cell death by activation of the GSDMD signalling axis in HLE-B3 hLECs. These results provide new insights into the exploitation of new candidates for the prevention of cataracts.

scholarly journals The effects of c-Src kinase on EMT signaling pathway in human lens epithelial cells associated with lens diseases

2019 ◽  
Author(s):  
Xing-Yu Li ◽  
Fang Wang ◽  
Mei-Xia Ren ◽  
Min-Juan Du ◽  
Jian Zhou
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Western Blot ◽  
Src Kinase ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Protein Levels ◽  
Human Lens Epithelial Cells ◽  
And Migration ◽  
E Cadherin

Abstract Background: The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors. Methods: Human lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src 418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-Src Y530F to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA, and vimentin were examined at 48 hours by RT-PCR and western blot. At 48 hours and 72 hours of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assay. Results: Activity of c-Src kinase, which causes the expression of p-Src 418 , was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-Src Y530F , the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src 418 increased. The expression of both E-cadherin and ZO-1 was suppressed, while the expressions of vimentin and α-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src 418 expression were knocked down. The expression of E-cadherin and ZO-1 increased, but the expression of vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced. Conclusions: The c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.

scholarly journals The effects of c-Src kinase on EMT signaling pathway in human lens epithelial cells associated with lens diseases

2019 ◽  
Author(s):  
Xing-Yu Li ◽  
Fang Wang ◽  
Mei-Xia Ren ◽  
Min-Juan Du ◽  
Jian Zhou
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Western Blot ◽  
Src Kinase ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Protein Levels ◽  
Human Lens Epithelial Cells ◽  
And Migration ◽  
E Cadherin

Abstract Background: The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors. Methods: Human lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src 418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-Src Y530F to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA, and vimentin were examined at 48 hours by RT-PCR and western blot. At 48 hours and 72 hours of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assay. Results: Activity of c-Src kinase, which causes the expression of p-Src 418 , was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-Src Y530F , the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src 418 increased. The expression of both E-cadherin and ZO-1 was suppressed, while the expressions of vimentin and α-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src 418 expression were knocked down. The expression of E-cadherin and ZO-1 increased, but the expression of vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced. Conclusions: The c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.

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