scholarly journals Deciphering the mechanoresponsive role of β-catenin in Keratoconus epithelium

2019 ◽  
Author(s):  
Chatterjee Amit ◽  
Prema Padmanabhan ◽  
Janakiraman Narayanan
Keyword(s):  
Cyclin D1 ◽  
Corneal Epithelium ◽  
Cell Adhesion Molecule ◽  
Corneal Stroma ◽  
Substrate Stiffness ◽  
Lipid Transfer ◽  
Immunofluorescence Studies ◽  
E Cadherin

AbstractKeratoconus (KC) a disease with established biomechanical instability of the corneal stroma, is an ideal platform to identify key proteins involved in mechanosensing. This study aims to investigate the possible role of β-catenin as mechanotransducer in KC epithelium. KC patients were graded as mild, moderate or severe using Amsler Krumeich classification. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyze the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, Rac123. Co-immunoprecipitation (Co-IP) of β-catenin followed by mass spectrometry of mild KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. We observed down regulation of E-cadherin, α-catenin, ZO1 and upregulation of Cyclin D1, α-actinin and RhoA in KC corneal epithelium. β-catenin Co-IP from mild KC epithelium identified new interacting partners such as StAR-related lipid transfer protein3, Dynamin-1-like protein, Cardiotrophin-1,Musculin, Basal cell adhesion molecule and Protocadherin Fat 1.β-catenin localization was altered in KC which was validatedin vitro, using control corneal epithelium grown on different substrate stiffness. β-catenin localization is dependent upon the elastic modulus of the substrate and acts as mechanotransducer by altering its interaction and regulating the barrier function in corneal epithelium.

scholarly journals Deciphering the mechanoresponsive role of β-catenin in keratoconus epithelium

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chatterjee Amit ◽  
Prema Padmanabhan ◽  
Janakiraman Narayanan
Keyword(s):  
Corneal Epithelium ◽  
Refractive Surgery ◽  
Structural Changes ◽  
Corneal Transplantation ◽  
Corneal Stroma ◽  
Corneal Dystrophy ◽  
Collagen Crosslinking ◽  
Immunofluorescence Studies ◽  
Ultrastructural Characteristics

AbstractKeratoconus (KC) is a corneal dystrophy characterized by progressive ectasia that leads to severe visual impairment and remains one of the leading indications for corneal transplantation. The etiology is believed to be multifactorial and alterations have been documented in the biomechanical, biochemical and ultrastructural characteristics of the cornea. While the exact site of disease origin is still debated, changes in the corneal epithelium are believed to occur even before the disease is clinically manifested. In this study we investigate the possible role of β-catenin as mechanotransducer in KC corneal epithelium. The sheets of corneal epithelium removed from keratoconic eyes when they underwent collagen crosslinking as a therapeutic procedure were used for this study. The healthy corneal epithelium of patients undergoing Laser Refractive Surgery for the correction of their refractive error, served as controls. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyse the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, and Rac123. Co-immunoprecipitation of β-catenin followed by mass spectrometry of KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. Histology data reported breaks in the Bowman’s layer in KC patients. We hypothesize that these breaks expose the epithelium to the keratoconic corneal stroma, which, is known to have a decreased elastic modulus and that β-catenin acts as a mechanotransducer that induces structural changes such as loss of polarity (Syntaxin3) and barrier function (ZO1) through membrane delocalization. The results of our study strongly suggest that β-catenin could be a putative mechanotransducer in KC epithelium, thus supporting our hypothesis.

scholarly journals Antiproliferative Effect of Androgen Receptor Inhibition in Mesenchymal Stem-Like Triple-Negative Breast Cancer

2016 ◽  
Vol 38 (3) ◽  
pp. 1003-1014 ◽  
Author(s):  
Aiyu Zhu ◽  
Yan Li ◽  
Wei Song ◽  
Yumei Xu ◽  
Fang Yang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Androgen Receptor ◽  
Cyclin D1 ◽  
Cancer Cells ◽  
Triple Negative Breast Cancer ◽  
Breast Cancer Cells ◽  
Triple Negative

Background/Aims: Androgen receptor (AR), a steroid hormone receptor, has recently emerged as prognostic and treatment-predictive marker in breast cancer. Previous studies have shown that AR is widely expressed in up to one-third of triple-negative breast cancer (TNBC). However, the role of AR in TNBC is still not fully understood, especially in mesenchymal stem-like (MSL) TNBC cells. Methods: MSL TNBC MDA-MB-231 and Hs578T breast cancer cells were exposed to various concentration of agonist 5-α-dihydrotestosterone (DHT) or nonsteroidal antagonist bicalutamide or untreated. The effects of AR on cell viability and apoptosis were determined by MTT assay, cell counting, flow cytometry analysis and protein expression of p53, p73, p21 and Cyclin D1 were analyzed by western blotting. The bindings of AR to p73 and p21 promoter were detected by ChIP assay. MDA-MB-231 cells were transplanted into nude mice and the tumor growth curves were determined and expression of AR, p73 and p21 were detected by Immunohistochemistry (IHC) staining after treatment of DHT or bicalutamide. Results: We demonstrate that AR agonist DHT induces MSL TNBC breast cancer cells proliferation and inhibits apoptosis in vitro. Similarly, activated AR significantly increases viability of MDA-MB-231 xenografts in vivo. On the contrary, AR antagonist, bicalutamide, causes apoptosis and exerts inhibitory effects on the growth of breast cancer. Moreover, DHT-dependent activation of AR involves regulation in the cell cycle related genes, including p73, p21 and Cyclin D1. Further investigations indicate the modulation of AR on p73 and p21 mediated by direct binding of AR to their promoters, and DHT could make these binding more effectively. Conclusions: Our study demonstrates the tumorigenesis role of AR and the inhibitory effect of bicalutamide in AR-positive MSL TNBC both in vitro and in vivo, suggesting that AR inhibition could be a potential therapeutic approach for AR-positive TNBC patients.

The extracellular matrix of the developing cornea: diversity, deposition and function*

Development ◽  
1988 ◽  
Vol 103 (Supplement) ◽  
pp. 195-205
Author(s):  
J. B. L. Bard ◽  
M. K. Bansal ◽  
A. S. A. Ross
Keyword(s):  
Extracellular Matrix ◽  
Chondroitin Sulphate ◽  
Corneal Stroma ◽  
Experimental System ◽  
Collagen I ◽  
And Function ◽  
20 Nm

This paper examines the role of the extracellular matrix (ECM) in the development of the cornea. After a brief summary of the corneal structure and ECM, we describe evidence suggesting that the differentiation of neural crest (NC) cells into endothelium and fibroblasts is under the control of ocular ECM. We then examine the role of collagen I in stromal morphogenesis by comparing normal corneas with those of homozygous Movl3 mice which do not make collagen I. We report that, in spite of this absence, the cellular morphology of the Movl3 eye is indistinguishable from that of the wild type. In the 16-day mutant stroma, however, the remaining collagens form small amounts of disorganized, thin fibrils rather than orthogonally organized 20 nm-diameter fibrils; a result implying that collagen I plays only a structural role and that its absence is not compensated for. It also suggests that, because these remaining collagens will not form the normal fibrils that they will in vitro, fibrillogenesis in the corneal stroma differs from that elsewhere. The latter part of the paper describes our current work on chick stromal deposition using corneal epithelia isolated with an intact basal lamina that lay down in vitro ∼3μm-thick stromas of organized fibrils similar to that seen in vivo. This experimental system has yielded two unexpected results. First, the amount of collagen and proteoglycans produced by such epithelia is not dependent on whether its substratum is collagenous and we therefore conclude that stromal production by the intact epithelium is more autonomous than hitherto thought. Second, chondroitin sulphate (CS), the predominant proteoglycan, appears to play no role in stromal morphogenesis: epithelia cultured in testicular hyaluronidase, which degrades CS, lay down stromas whose organization and fibrildiameter distribution are indistinguishable from controls. One possible role for CS, however, is as a lubricant which facilitates corneal growth: it could allow fibrils to move over one another without deforming their orthogonal organization. Finally, we have examined the processes of fibrillogenesis in the corneal stroma and conclude that they are different from those elsewhere in the embryo and in vitro, perhaps because there is in the primary stroma an unidentified, highly hydrated ECM macromolecule that embeds the fibrils and that may mediate their morphogenesis.

scholarly journals Role of PTH1R internalization in osteoblasts and bone mass using a phosphorylation-deficient knock-in mouse model

2010 ◽  
Vol 207 (3) ◽  
pp. 355-365 ◽  
Author(s):  
Nabanita S Datta ◽  
Tareq A Samra ◽  
Chandrika D Mahalingam ◽  
Tanuka Datta ◽  
Abdul B Abou-Samra
Keyword(s):  
Mouse Model ◽  
Cyclin D1 ◽  
Intracellular Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Bone Homeostasis ◽  
Low Calcium Diet ◽  
High Calcium ◽  
The Impact

Phosphorylation, internalization, and desensitization of G protein-coupled receptors, such as the parathyroid hormone (PTH) and PTH-related peptide (PTHrP) receptor (PTH1R), are well characterized and known to regulate the cellular responsiveness in vitro. However, the role of PTH1R receptor phosphorylation in bone formation and osteoblast functions has not yet been elucidated. In previous studies, we demonstrated impaired internalization and sustained cAMP stimulation of a phosphorylation-deficient (pd) PTH1R in vitro, and exaggerated cAMP and calcemic responses to s.c. PTH infusion in pdPTH1R knock-in mouse model. In this study, we examined the impact of impaired PTH1R phosphorylation on the skeletal phenotype of mice maintained on normal, low, and high calcium diets. The low calcium diet moderately reduced (P<0.05) bone volume and trabecular number, and increased trabecular spacing in both wild-type (WT) and pd mice. The effects, however, seem to be less pronounced in the female pd compared to WT mice. In primary calvarial osteoblasts isolated from 2-week-old pd or WT mice, PTH and PTHrP decreased phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2), a member of mitogen-activated protein kinase, and cyclin D1, a G1/S phase cyclin, in vitro. In contrast to WT osteoblasts, down-regulation of cyclin D1 was sustained for longer periods of time in osteoblasts isolated from the pd mice. Our results suggest that adaptive responses of intracellular signaling pathways in the pd mice may be important for maintaining bone homeostasis.

Combined Epigenetic and Immunotherapy For Newly Diagnosed Mantle Cell Lymphoma: Correlative Studies Suggest The Importance Of Enhanced ADCC, Mechanisms of Resistance and Cyclin D1 Nuclear Localization Genotype

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3063-3063
Author(s):  
Kamal Sharma ◽  
Violetta V. Leshchenko ◽  
Zainul Hasanali ◽  
August Stuart ◽  
Sara Shimko ◽  
...  
Keyword(s):  
Complete Remission ◽  
Cyclin D1 ◽  
Nuclear Localization ◽  
Cell Lymphoma ◽  
Newly Diagnosed ◽  
Mantle Cell ◽  
Correlative Studies

Abstract Previously, we reported that epigenetic therapy with cladribine, SAHA, and rituximab (SCR) for newly diagnosed mantle cell lymphoma was remarkably effective, with 100% overall response rate, 85-90% CR rate, and durable responses( Hasanali, AACR,2013 LBA140). Over 40 patients now have been enrolled with all patients completing therapy. Final response and CR rates will be reported at the meeting. This abstract will focus on the correlative studies performed as part of this trial. Cladribine, a purine analog with reported epigenetic activity was shown here by HELP assays to inhibit DNA methylation in vivo in 6 patients with leukemic MCL. Similar activity was also observed in two MCL and two CLL patients treated with cladribine without vorinostat off trial, suggesting cladribine is a DNA hypomethylating agent. Due to cladribine's ability to inhibit the enzyme SAH hydrolase and thus inhibit the donation of methyl groups by S-adenosyl methionine (SAM), we assayed the ability of cladibine to inhibit histone methylation in vitro by Western blot analysis and in vitro assays of histone methyltransferase (HMT) activity on H3lys9 and H3lys27. Both assays demonstrated inhibition of methylated histones (Western) and HMT activity using MCL cell cells and nuclear extract at concentrations of cladribine in the 10-20 um range, higher than the in vivo concentration of 10-20 nm. These observations could be due to the lack of sensitivity of these assays, and more sensitive assays are in development. Studies to help elucidate the mechanism of action of synergy of epigenetic drugs with the monoclonal antibody rituximab were performed. Using cells from patients with leukemic MCL treated with SCR, we assayed for characteristic changes of apoptosis using Western blotting and TUNEL assays. None were detected. We were unable to observe complement mediated cytoxicity in vitro using human serum and rituximab with added cladribine or vorinostat. With ADCC being the primary mechanism of presumed combined epigenetic and rituximab synergy, we investigated ADCC further. CD137 transcriptional upregulation was seen in several but not all treated patients, and some patients showed up regulation of perforin and granzyme mRNA by QRTPCR. An NK cell line, NKL, showed transcriptional upregulation of CD137 after treatment with cladribine and vorinostat. A polymorphism at an intron-exon junction effects the nuclear localization of cyclin D1 by removing a nuclear export signal. Although there is published evidence supporting the role of nuclear cyclin D1 in increased oncogenesis, the role of this polymorphism in MCL remains controversial. Samples from peripheral blood of patients on trial were genotyped at the cyclin D1 locus as AA, AG, or GG, with the A allele being the loss of function allele. The presence of the A allele strongly correlated with the blastic phenotype and the lack of complete remission after SCR therapy, with both being statistically significant (table 1). Immunofluorescent studies with cyclin D1 antibodies showed nuclear and cytoplasmic localization as predicted in patients with the AA and GG genotypes (Fig 1). The heterozygotes are under investigation and will be reported. The mechanism of resistance to SCR was studied in a patient with blastic, leukemic MCL. A 63 male achieved complete remission after two cycles of SCR. Subsequently, he developed neurologic symptoms and was found to have CNS disease. At autopsy, CD20+ disease was found in his CNS and CD20- disease was found systemically. A cell line was established from his peripheral blood that showed significantly reduced levels of CD20 mRNA. Treatment of these cells with a variety of epigenetic drugs was unable to upregulate CD20 mRNA. These cells have been in continuous culture for over 1 year and continue to show diminished levels of CD20 mRNA and protein. Epigenetic changes at the promoter are being studied by chromatin immunoprecipation (ChiP) assays. Disclosures: No relevant conflicts of interest to declare.

Surface Modified Poly(vinyl alcohol) Nanofiber for the Artificial Corneal Stroma

2007 ◽  
Vol 342-343 ◽  
pp. 209-212 ◽  
Author(s):  
Hisatoshi Kobayashi
Keyword(s):  
Corneal Epithelium ◽  
Vinyl Alcohol ◽  
Type I Collagen ◽  
Corneal Stroma ◽  
Light Transmittance ◽  
Poly Vinyl Alcohol ◽  
Type I ◽  
Nanofiber Sheets

Previously we have found that the immobilization of Type I collagen on the poly(vinyl alcohol)(PVA) hydrogel disc was effective in supporting adhesion and growth of the corneal epithelium and stromal cell in vitro. But the durability of the produced corneal epithelium layer in vivo has some problem. We hypothesized the cell construction force is much stronger than the force of the cell adhesion on the flat modified PVA surfaces. Therefore the improvement of mechanical anchoring force between the substrate and formed corneal cell layer maybe become one of the solving methods. In this study, we prepared the PVA nanofiber mat by using the electrospinning method and the surface modification of the PVA nanofiber was studied to improve the durability of the corneal epithelium layer. The collagen-immobilized PVA nanofiber sheets could support the adhesion and proliferation of rabbit corneal epithelial cells. And the stratified corneal epithelium structure was observed on the PVA nanofiber sheets when the epithelium was co-cultured with rabbit corneal stromal cells. It means that the corneal epithelium was well differentiated on the collagen immobilized PVA nanofiber sheet. The stability of the corneal epithelium layer on the PVA was dramatically improved; the stratified epithelium layer was kept for two weeks after the differentiation introduction, totally after one month. A light transmittance of these materials is not yet enough. Further study to improve the transmission of light, is required.

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