scholarly journals Cigarette Smoke-Induced Lymphoid Neogenesis in COPD Involves IL-17/RANKL Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Jing Xiong ◽  
Lu Zhou ◽  
Jieyu Tian ◽  
Xia Yang ◽  
Yunsong Li ◽  
...  
Keyword(s):  
B Cells ◽  
Cigarette Smoke ◽  
Molecular Mechanisms ◽  
B Lymphocyte ◽  
Nuclear Factor Κb ◽  
Lymphoid Follicle ◽  
Lymphoid Follicles ◽  
Receptor Activator

IL-17 is critical in lung lymphoid neogenesis in COPD, but the cellular and molecular mechanisms remain to be elucidated. Receptor activator of nuclear factor-κB ligand (RANKL) functions in lymphoid follicle formation in other organs, whether it is involved in IL-17A–dependent lymphoid neogenesis in COPD is unknown. To elucidate the expression and functional role of IL-17A/RANKL pathway in COPD. We first quantified and localized RANKL, its receptor RANK and IL-17A in lungs of patients with COPD, smokers and non-smokers. Next, IL-17A−/− and wild-type (WT) mice were exposed to air or cigarette smoke (CS) for 24 weeks, and lung lymphoid follicles and RANKL-RANK expression were measured. Lastly, we studied the in vitro biological function of RANKL pertaining to lymphoid neogenesis. We found that the expressions of RANKL-RANK and IL-17A, together with lymphoid follicles, were increased in lung tissues from patients with COPD. In WT mice exposed to CS, RANKL-RANK expressions were prominent in lung lymphoid follicles, which were absent in IL-17A−/− mice exposed to CS. In the lymphoid follicles, RANKL+ cells were identified mostly as B cells and RANK was localized in dendritic cells (DCs). In vitro IL-17A increased the expressions of RANKL in B cells and RANK in DCs, which in turn responded to RANKL stimulation by upregulation of CXCL13. Altogether, these results suggest that B lymphocyte RANKL pathway is involved in IL-17A–dependent lymphoid neogenesis in COPD.

scholarly journals The Role of miR-21 in Osteoblasts–Osteoclasts Coupling In Vitro

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 479 ◽  
Author(s):  
Agnieszka Smieszek ◽  
Klaudia Marcinkowska ◽  
Ariadna Pielok ◽  
Mateusz Sikora ◽  
Lukas Valihrach ◽  
...  
Keyword(s):  
Cathepsin K ◽  
Nuclear Factor Κb ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Nuclear Factor ◽  
Paracrine Signaling ◽  
Receptor Activator ◽  
The Impact

MiR-21 is being gradually more and more recognized as a molecule regulating bone tissue homeostasis. However, its function is not fully understood due to the dual role of miR-21 on bone-forming and bone-resorbing cells. In this study, we investigated the impact of miR-21 inhibition on pre-osteoblastic cells differentiation and paracrine signaling towards pre-osteoclasts using indirect co-culture model of mouse pre-osteoblast (MC3T3) and pre-osteoclast (4B12) cell lines. The inhibition of miR-21 in MC3T3 cells (MC3T3inh21) modulated expression of genes encoding osteogenic markers including collagen type I (Coll-1), osteocalcin (Ocl), osteopontin (Opn), and runt-related transcription factor 2 (Runx-2). Inhibition of miR-21 in osteogenic cultures of MC3T3 also inflected the synthesis of OPN protein which is essential for proper mineralization of extracellular matrix (ECM) and anchoring osteoclasts to the bones. Furthermore, it was shown that in osteoblasts miR-21 regulates expression of factors that are vital for survival of pre-osteoclast, such as receptor activator of nuclear factor κB ligand (RANKL). The pre-osteoclast cultured with MC3T3inh21 cells was characterized by lowered expression of several markers associated with osteoclasts’ differentiation, foremost tartrate-resistant acid phosphatase (Trap) but also receptor activator of nuclear factor-κB ligand (Rank), cathepsin K (Ctsk), carbonic anhydrase II (CaII), and matrix metalloproteinase (Mmp-9). Collectively, our data indicate that the inhibition of miR-21 in MC3T3 cells impairs the differentiation and ECM mineralization as well as influences paracrine signaling leading to decreased viability of pre-osteoclasts.

scholarly journals Nox4 Promotes RANKL-Induced Autophagy and Osteoclastogenesis via Activating ROS/PERK/eIF-2α/ATF4 Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Sun ◽  
Wugui Chen ◽  
Songtao Li ◽  
Sizhen Yang ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Nuclear Factor Κb ◽  
Potential Therapeutic Target ◽  
Unfolded Protein ◽  
Nadph Oxidase 4 ◽  
Receptor Activator ◽  
Ros Scavenger ◽  
Protein Response

Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Via conducting a series of biochemical experiments with in vitro cell lines, this study investigated the role and mechanism of NADPH oxidase 4 (Nox4) in RANKL-induced autophagy and osteoclastogenesis. In the current study, we found that RANKL dramatically induced autophagy and osteoclastogenesis, inhibition of autophagy with chloroquine (CQ) markedly attenuates RANKL-induced osteoclastogenesis. Interestingly, we found that the protein level of Nox4 was remarkably upregulated by RANKL treatment. Inhibition of Nox4 by 5-O-methyl quercetin or knockdown of Nox4 with specific shRNA markedly attenuated RANKL-induced autophagy and osteoclastogenesis. Furthermore, we found that Nox4 stimulated the production of nonmitochondrial reactive oxygen species (ROS), activating the critical unfolded protein response (UPR)-related signaling pathway PERK/eIF-2α/ATF4, leading to RANKL-induced autophagy and osteoclastogenesis. Blocking the activation of PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS scavenger (NAC) or PERK inhibitor (GSK2606414) significantly inhibited autophagy during RANKL-induced osteoclastogenesis. Collectively, this study reveals that Nox4 promotes RANKL-induced autophagy and osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 pathway, suggesting that the pathway may be a novel potential therapeutic target for osteoclastogenesis-related disease.

scholarly journals Microfluidic tumor-on-a-chip model to evaluate the role of tumor environmental stress on NK cell exhaustion

2021 ◽  
Vol 7 (8) ◽  
pp. eabc2331 ◽  
Author(s):  
Jose M. Ayuso ◽  
Shujah Rehman ◽  
Maria Virumbrales-Munoz ◽  
Patrick H. McMinn ◽  
Peter Geiger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Nk Cell ◽  
Checkpoint Inhibitors ◽  
Waste Product ◽  
Extended Period ◽  
Cell Exhaustion

Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.

scholarly journals Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

2021 ◽  
Vol 22 (3) ◽  
pp. 1163
Author(s):  
Gaia Palmini ◽  
Cecilia Romagnoli ◽  
Simone Donati ◽  
Roberto Zonefrati ◽  
Gianna Galli ◽  
...  
Keyword(s):  
Cell Line ◽  
Molecular Mechanisms ◽  
Osteogenic Sarcoma ◽  
Cancer Cell Line ◽  
Microscopic Features ◽  
In Vitro Establishment ◽  
Profile Characteristic ◽  
First Time

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

scholarly journals Pivotal role of MUC1 glycosylation by cigarette smoke in modulating disruption of airway adherens junctions in vitro

2014 ◽  
Vol 234 (1) ◽  
pp. 60-73 ◽  
Author(s):  
Lili Zhang ◽  
Marianne Gallup ◽  
Lorna Zlock ◽  
Yu Ting Feeling Chen ◽  
Walter E Finkbeiner ◽  
...  
Keyword(s):  
Cigarette Smoke ◽  
Adherens Junctions ◽  
Pivotal Role

scholarly journals Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

2020 ◽  
Vol 10 (1) ◽  
pp. 78
Author(s):  
April Nettesheim ◽  
Myoung Sup Shim ◽  
Angela Dixon ◽  
Urmimala Raychaudhuri ◽  
Haiyan Gong ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Ecm Remodeling ◽  
Trabecular Meshwork Cells ◽  
Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

scholarly journals Low Intensity Shockwave Treatment Modulates Macrophage Functions Beneficial to Healing Chronic Wounds

2021 ◽  
Vol 22 (15) ◽  
pp. 7844
Author(s):  
Jason S. Holsapple ◽  
Ben Cooper ◽  
Susan H. Berry ◽  
Aleksandra Staniszewska ◽  
Bruce M. Dickson ◽  
...  
Keyword(s):  
Macrophage Activation ◽  
Molecular Mechanisms ◽  
Chronic Wounds ◽  
Immunohistochemical Analysis ◽  
Therapeutic Effects ◽  
Gene Expressions ◽  
Extracorporeal Shock Wave

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150–500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

scholarly journals Porphyromonas gingivalis Infection-Associated Periodontal Bone Resorption Is Dependent on Receptor Activator of NF-κB Ligand

2013 ◽  
Vol 81 (5) ◽  
pp. 1502-1509 ◽  
Author(s):  
Xiaozhe Han ◽  
Xiaoping Lin ◽  
Xiaoqian Yu ◽  
Jiang Lin ◽  
Toshihisa Kawai ◽  
...  
Keyword(s):  
B Cells ◽  
Bone Resorption ◽  
Fusion Protein ◽  
Porphyromonas Gingivalis ◽  
Nuclear Factor Κb ◽  
Infected Group ◽  
T And B Cells ◽  
Content Type ◽  
Receptor Activator ◽  
Control Fusion

ABSTRACTPorphyromonas gingivalisis one of the oral microorganisms associated with human chronic periodontitis. The purpose of this study is to determine the role of the receptor activator of nuclear factor-κB ligand (RANKL) inP. gingivalisinfection-associated periodontal bone resorption. Inbred female Rowett rats were infected orally on four consecutive days (days 0 to 3) with 1 × 109P. gingivalisbacteria (strain ATCC 33277). Separate groups of rats also received an injection of anti-RANKL antibody, osteoprotegerin fusion protein (OPG-Fc), or a control fusion protein (L6-Fc) into gingival papillae in addition toP. gingivalisinfection. Robust serum IgG and salivary IgA antibody (P< 0.01) and T cell proliferation (P< 0.05) responses toP. gingivaliswere detected at day 7 and peaked at day 28 inP. gingivalis-infected rats. Both the concentration of soluble RANKL (sRANKL) in rat gingival tissues (P< 0.01) and periodontal bone resorption (P< 0.05) were significantly elevated at day 28 in theP. gingivalis-infected group compared to levels in the uninfected group. Correspondingly, RANKL-expressing T and B cells in rat gingival tissues were significantly increased at day 28 in theP. gingivalis-infected group compared to the levels in the uninfected group (P< 0.01). Injection of anti-RANKL antibody (P< 0.05) or OPG-Fc (P< 0.01), but not L6-Fc, into rat gingival papillae afterP. gingivalisinfection resulted in significantly reduced periodontal bone resorption. This study suggests thatP. gingivalisinfection-associated periodontal bone resorption is RANKL dependent and is accompanied by increased local infiltration of RANKL-expressing T and B cells.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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