scholarly journals Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy

2021 ◽  
Author(s):  
Marta Słoniecka ◽  
André Vicente ◽  
Berit Byström ◽  
Fátima Pedrosa Domellöf
Keyword(s):  
Protein Expression ◽  
Signaling Pathways ◽  
Cell Fate ◽  
Expression Patterns ◽  
Pax6 Gene ◽  
Cas9 Nuclease ◽  
Extracellular Matrix Components ◽  
Gene And Protein Expression ◽  
Human Keratocytes

ABSTRACTPURPOSETo establish an in vitro model of aniridia-related keratopathy (ARK) using CRISPR/Cas9 engineered human keratocytes with mutations in the PAX6 gene, and to study the Notch Homolog 1, Translocation-Associated (Notch1), sonic hedgehog (SHH), mammalian target of rapamycin (mTOR), and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes.METHODSPrimary human keratocytes were isolated from healthy corneas. Keratocytes were transduced with Cas9 lentiviral particles in order to create cells stably expressing Cas9 nuclease. Lentiviral particles carrying PAX6 sgRNA were transduced into the Cas9 keratocytes creating mutants. Analysis of signaling pathways was assessed by RT-qPCR for gene expression and western blot for protein expression.RESULTSHuman keratocytes stably expressing Cas9 nuclease were created. Keratocytes carrying PAX6 gene mutation were successfully generated. PAX6 mutant keratocytes showed modified expression patterns of extracellular matrix components such as collagens and fibrotic markers. Analysis of the Notch1, SHH, mTOR, and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes revealed altered gene and protein expression of the key players involved in these pathways.CONCLUSIONSA properly functioning PAX6 gene in keratocytes is crucial for the regulation of signaling pathways important for cell fate determination, proliferation, and inflammation. Pax6 mutation in the in vitro settings leads to changes in these pathways which resemble those found in corneas of patients with ARK.

scholarly journals Identification of pluripotent cells in bovine uterus: in situ and in vitro studies

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 317-327 ◽  
Author(s):  
Martyna Łupicka ◽  
Gabriel Bodek ◽  
Nahum Shpigel ◽  
Ehud Elnekave ◽  
Anna J Korzekwa
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Mrna Expression ◽  
Uterine Horn ◽  
Uterine Tissue ◽  
Pluripotent Cells ◽  
Flow Cytometry Assay ◽  
Myometrial Cells ◽  
Gene And Protein Expression

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8–10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

scholarly journals Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision

BioTechniques ◽  
2004 ◽  
Vol 36 (6) ◽  
pp. 1030-1037 ◽  
Author(s):  
Annika Spruessel ◽  
Garnet Steimann ◽  
Mira Jung ◽  
Sung A. Lee ◽  
Theresa Carr ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Patterns ◽  
Tumor Excision ◽  
Ischemia Time ◽  
Tissue Ischemia ◽  
Gene And Protein Expression

Feedback regulation is central to Delta-Notch signalling required for Drosophila wing vein morphogenesis

Development ◽  
1997 ◽  
Vol 124 (17) ◽  
pp. 3283-3291 ◽  
Author(s):  
S.S. Huppert ◽  
T.L. Jacobsen ◽  
M.A. Muskavitch
Keyword(s):  
Protein Expression ◽  
Cell Fate ◽  
Expression Patterns ◽  
Feedback Regulation ◽  
Notch Signalling ◽  
Notch Receptor ◽  
Cell Fates ◽  
Wing Vein ◽  
Drosophila Wing ◽  
Puparium Formation

Delta and Notch are required for partitioning of vein and intervein cell fates within the provein during Drosophila metamorphosis. We find that partitioning of these fates is dependent on Delta-mediated signalling from 22 to 30 hours after puparium formation at 25 degrees C. Within the provein, Delta is expressed more highly in central provein cells (presumptive vein cells) and Notch is expressed more highly in lateral provein cells (presumptive intervein cells). Accumulation of Notch in presumptive intervein cells is dependent on Delta signalling activity in presumptive vein cells and constitutive Notch receptor activity represses Delta accumulation in presumptive vein cells. When Delta protein expression is elevated ectopically in presumptive intervein cells, complementary Delta and Notch expression patterns in provein cells are reversed, and vein loss occurs because central provein cells are unable to stably adopt the vein cell fate. Our findings imply that Delta-Notch signalling exerts feedback regulation on Delta and Notch expression during metamorphic wing vein development, and that the resultant asymmetries in Delta and Notch expression underlie the proper specification of vein and intervein cell fates within the provein.

scholarly journals Estrogen Signaling Drives Ciliogenesis in Human Endometrial Organoids

Endocrinology ◽  
2019 ◽  
Vol 160 (10) ◽  
pp. 2282-2297 ◽  
Author(s):  
Sandra Haider ◽  
Magdalena Gamperl ◽  
Thomas R Burkard ◽  
Victoria Kunihs ◽  
Ulrich Kaindl ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Expression Patterns ◽  
Estrogen Signaling ◽  
Ciliated Cells ◽  
Cell Fate Decisions ◽  
Primary Driver ◽  
Endometrial Epithelium ◽  
Cyclic Changes

Abstract The human endometrium is the inner lining of the uterus consisting of stromal and epithelial (secretory and ciliated) cells. It undergoes a hormonally regulated monthly cycle of growth, differentiation, and desquamation. However, how these cyclic changes control the balance between secretory and ciliated cells remains unclear. Here, we established endometrial organoids to investigate the estrogen (E2)-driven control of cell fate decisions in human endometrial epithelium. We demonstrate that they preserve the structure, expression patterns, secretory properties, and E2 responsiveness of their tissue of origin. Next, we show that the induction of ciliated cells is orchestrated by the coordinated action of E2 and NOTCH signaling. Although E2 is the primary driver, inhibition of NOTCH signaling provides a permissive environment. However, inhibition of NOTCH alone is not sufficient to trigger ciliogenesis. Overall, we provide insights into endometrial biology and propose endometrial organoids as a robust and powerful model for studying ciliogenesis in vitro.

scholarly journals Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Neety Sahu ◽  
Gaurav Budhiraja ◽  
Anuradha Subramanian
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Signaling Pathways ◽  
Mesenchymal Stromal Cells ◽  
Nuclear Localization ◽  
Stromal Cells ◽  
Actin Reorganization ◽  
Low Intensity ◽  
Gene And Protein Expression ◽  
Low Intensity Ultrasound

Abstract Background Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

scholarly journals Extracellular SPARC cooperates with TGF-β signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts

Rheumatology ◽  
2019 ◽  
Vol 59 (9) ◽  
pp. 2258-2263 ◽  
Author(s):  
Tiago Carvalheiro ◽  
Beatriz Malvar Fernández ◽  
Andrea Ottria ◽  
Barbara Giovannone ◽  
Wioleta Marut ◽  
...  
Keyword(s):  
Protein Expression ◽  
Therapeutic Target ◽  
Dermal Fibroblasts ◽  
Dependent Manner ◽  
Healthy Controls ◽  
Multiple Organs ◽  
Extracellular Matrix Components ◽  
Mrna And Protein Expression

Abstract Objectives SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. Methods Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-β1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. Results Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-β and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-β signalling. Conclusion These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

scholarly journals Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions

Toxins ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 404 ◽  
Author(s):  
Rayana Maciel ◽  
Regiane Cunha ◽  
Valentina Busato ◽  
Célia Franco ◽  
Paulo Gregório ◽  
...  
Keyword(s):  
Protein Expression ◽  
Intercellular Junctions ◽  
Serum Levels ◽  
Endothelial Damage ◽  
Cell Junctions ◽  
Cell Junction ◽  
Uremic Serum ◽  
Gene And Protein Expression ◽  
The Impact

Endothelial dysfunction in uremia can result in cell-to-cell junction loss and increased permeability, contributing to cardiovascular diseases (CVD) development. This study evaluated the impact of the uremic milieu on endothelial morphology and cell junction’s proteins. We evaluated (i) serum levels of inflammatory biomarkers in a cohort of chronic kidney disease (CKD) patients and the expression of VE-cadherin and Zonula Occludens-1 (ZO-1) junction proteins on endothelial cells (ECs) of arteries removed from CKD patients during renal transplant; (ii) ECs morphology in vitro under different uremic conditions, and (iii) the impact of uremic toxins p-cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) as well as of total uremic serum on VE-cadherin and ZO-1 gene and protein expression in cultured ECs. We found that the uremic arteries had lost their intact and continuous endothelial morphology, with a reduction in VE-cadherin and ZO-1 expression. In cultured ECs, both VE-cadherin and ZO-1 protein expression decreased, mainly after exposure to Pi and uremic serum groups. VE-cadherin mRNA expression was reduced while ZO-1 was increased after exposure to PCS, IS, Pi, and uremic serum. Our findings show that uremia alters cell-to-cell junctions leading to an increased endothelial damage. This gives a new perspective regarding the pathophysiological role of uremia in intercellular junctions and opens new avenues to improve cardiovascular outcomes in CKD patients.

scholarly journals Dehydroepiandrosterone Prevents H2O2-Induced BRL-3A Cell Oxidative Damage through Activation of PI3K/Akt Pathways rather than MAPK Pathways

2019 ◽  
Vol 2019 ◽  
pp. 1-14
Author(s):  
Longlong Li ◽  
Yao Yao ◽  
Zhihao Jiang ◽  
Jinlong Zhao ◽  
Ji Cao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
Signaling Pathways ◽  
Hormone Receptors ◽  
Mapk Signaling ◽  
Ros Generation ◽  
Protective Effects ◽  
Pi3k Inhibitor Ly294002 ◽  
Beneficial Effects

Dehydroepiandrosterone (DHEA) is a popular dietary supplement that has well-known benefits in animals and humans, but there is not enough information about the mechanisms underlying its effects. The present study aimed at investigating these mechanisms through in vitro experiments on the effects of DHEA on rat liver BRL-3A cells exposed to oxidative stress through H2O2. The findings showed that DHEA increased the antioxidant enzyme activity, decreased ROS generation, and inhibited apoptosis in H2O2-treated cells. These effects of DHEA were not observed when the cells were pretreated with known antagonists of sex hormones (Trilostane, Flutamide, or Fulvestrant). Furthermore, treatment with estradiol and testosterone did not have the same protective effects as DHEA. Thus, the beneficial effects of DHEA were associated with mechanisms that were independent of steroid hormone pathways. With regard to the mechanism underlying the antiapoptotic effect of DHEA, pretreatment with DHEA was found to induce a significant decrease in the protein expression of Bax and caspase-3 and a significant increase in the protein expression of PI3K and p-Akt in H2O2-treated BRL-3A cells. These effects of DHEA were abolished when the cells were pretreated with the PI3K inhibitor LY294002. No changes were observed on the p-ERK1/2, p-p38, and p-JNK protein levels in H2O2-induced BRL-3A cells pretreated with DHEA. In conclusion, our data demonstrate that DHEA protects BRL-3A cells against H2O2-induced oxidative stress and apoptosis through mechanisms that do not involve its biotransformation into steroid hormones or the activation of sex hormone receptors. Importantly, the protective effect of DHEA on BRL-3A cells was mainly associated with PI3K/Akt signaling pathways, rather than MAPK signaling pathways.

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