scholarly journals Commitment to differentiation and expression of early differentiation markers in murine keratinocytes in vitro are regulated independently of extracellular calcium concentrations.

1993 ◽  
Vol 123 (4) ◽  
pp. 909-919 ◽  
Author(s):  
V Drozdoff ◽  
W J Pledger
Keyword(s):  
Structural Changes ◽  
Solid Medium ◽  
Extracellular Calcium ◽  
Keratinocyte Differentiation ◽  
Differentiation Markers ◽  
Primary Mouse ◽  
Semi Solid ◽  
Mouse Keratinocytes

In the epidermis, one of the earliest characterized events in keratinocyte differentiation is the coordinate induction of a pair of keratins specifically expressed in suprabasal cells, keratin 1 (K1) and keratin 10 (K10). Both in vivo and in vitro, extracellular calcium is necessary for several biochemical and structural changes during keratinocyte differentiation. However, it has been unclear if calcium serves as a differentiation signal in keratinocytes. In these studies, expression of suprabasal keratin mRNA and protein is used to test whether the initial differentiation of primary mouse keratinocytes in vitro is dependent on changes in the concentration of extracellular calcium. K1 mRNA was expressed at low levels in cultures of keratinocytes growing on plastic in 0.05 mM calcium but in attached cells was not further induced by increases in the concentration of extracellular calcium. Suspension of the keratinocytes into semi-solid medium induced a rapid and substantial increase in both expression of K1 mRNA and in the percentage of cells expressing suprabasal keratin proteins. The induction was unaffected by the concentration of calcium in the semi-solid medium and could not be enhanced by exposing attached cells to higher calcium before suspension. The induction of K1 mRNA could be inhibited by exposure of the keratinocytes to either EGF or fibronectin. These results suggest that commitment of mouse keratinocytes to terminal differentiation is independent of extracellular calcium and may be regulated primarily by extracellular factors other than calcium.

scholarly journals Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Chandrama Shrestha ◽  
Yuanyuan Tang ◽  
Hong Fan ◽  
Lusha Li ◽  
Qin Zeng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Second Messengers ◽  
Human Keratinocytes ◽  
Extracellular Calcium ◽  
Keratinocyte Differentiation ◽  
Differentiation Markers ◽  
Calcium Stimulation ◽  
E Cadherin

Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1αis recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1αand the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1αactivation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1αand induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1αactivation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1αcomplex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1αcomplex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation.

scholarly journals Three Constituents of Moringa oleifera Seeds Regulate Expression of Th17-Relevant Cytokines and Ameliorate TPA-Induced Psoriasis-Like Skin Lesions in Mice

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3256 ◽  
Author(s):  
Nuan Ma ◽  
Qin Tang ◽  
Wan-Ting Wu ◽  
Xin-An Huang ◽  
Qin Xu ◽  
...  
Keyword(s):  
Moringa Oleifera ◽  
Skin Diseases ◽  
Critical Role ◽  
Folk Medicine ◽  
Skin Lesions ◽  
Keratinocyte Differentiation ◽  
Therapeutic Application ◽  
Differentiation Markers

As a folk medicine, Moringa oleifera L. is used effectively to treat inflammatory conditions and skin diseases. However, its mechanism of action is not well understood, limiting its medical use. We isolated and identified three compounds, namely niazirin, marumoside A and sitosterol-3-O-β-d-glucoside, from the seeds of Moringa oleifera, and studied their effects on the expression of Th17-relevant cytokines (IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19) using lipopolysaccharide-stimulated THP-1 cells. Additionally, as Th17 plays a critical role in the pathogenesis of psoriasis, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced psoriasis-like skin lesion mouse model to study their potential therapeutic application in vivo. The compounds suppressed the expression of IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19 in vitro, and in vivo they ameliorated psoriasis-like skin lesions, decreased IL-17A mRNA expression, and increased the expression of keratinocyte differentiation markers. To our knowledge, this is the first report regarding the mechanism and therapeutic application of Moringa oleifera seeds to treat psoriasis-like lesions in vivo.

scholarly journals Involvement of HECTD1 in LPS-induced astrocyte activation via σ-1R-JNK/p38-FOXJ2 axis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ying Tang ◽  
Mengchun Zhou ◽  
Rongrong Huang ◽  
Ling Shen ◽  
Li Yang ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Astrocyte Activation ◽  
Hect Domain ◽  
P38 Inhibitor ◽  
Ubiquitin Protein Ligase ◽  
Primary Mouse ◽  
Lps Treatment

Abstract Background Astrocytes participate in innate inflammatory responses within the mammalian central nervous system (CNS). HECT domain E3 ubiquitin protein ligase 1 (HECTD1) functions during microglial activation, suggesting a connection with neuroinflammation. However, the potential role of HECTD1 in astrocytes remains largely unknown. Results Here, we demonstrated that HECTD1 was upregulated in primary mouse astrocytes after 100 ng/ml lipopolysaccharide (LPS) treatment. Genetic knockdown of HECTD1 in vitro or astrocyte-specific knockdown of HECTD1 in vivo suppressed LPS-induced astrocyte activation, whereas overexpression of HECTD1 in vitro facilitated LPS-induced astrocyte activation. Mechanistically, we established that LPS activated σ-1R-JNK/p38 pathway, and σ-1R antagonist BD1047, JNK inhibitor SP600125, or p38 inhibitor SB203580 reversed LPS-induced expression of HECTD1, thus restored LPS-induced astrocyte activation. In addition, FOXJ2 functioned as a transcription factor of HECTD1, and pretreatment of primary mouse astrocytes with BD1047, SB203580, and SP600125 significantly inhibited LPS-mediated translocation of FOXJ2 into the nucleus. Conclusions Overall, our present findings suggest that HECTD1 participates in LPS-induced astrocyte activation by activation of σ-1R-JNK/p38-FOXJ2 pathway and provide a potential therapeutic strategy for neuroinflammation induced by LPS or any other neuroinflammatory disorders.

scholarly journals Interaction of Lactoferrin with Unsaturated Fatty Acids: In Vitro and In Vivo Study of Human Lactoferrin/Oleic Acid Complex Cytotoxicity

Materials ◽  
2021 ◽  
Vol 14 (7) ◽  
pp. 1602
Author(s):  
Anna Elizarova ◽  
Alexey Sokolov ◽  
Valeria Kostevich ◽  
Ekaterina Kisseleva ◽  
Evgeny Zelenskiy ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Structural Changes ◽  
Unsaturated Fatty Acids ◽  
Structural Features ◽  
Control Group ◽  
Acid Complex ◽  
Hepatoma 22A ◽  
Constitutive Parameters

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

scholarly journals Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 320
Author(s):  
Thaís Pereira da Silva ◽  
Fernando Jacomini de Castro ◽  
Larissa Vuitika ◽  
Nayanne Louise Costacurta Polli ◽  
Bruno César Antunes ◽  
...  
Keyword(s):  
Structural Changes ◽  
Capillary Permeability ◽  
Structural Data ◽  
Histopathological Analysis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Antigenic Properties ◽  
Harmful Effects

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

Peptide Blocking Self-Polymerization of Extracellular Calcium-Sensing Receptor Attenuates Hypoxia-Induced Pulmonary Hypertension

Hypertension ◽  
2021 ◽  
Author(s):  
Rui Xiao ◽  
Shengquan Luo ◽  
Ting Zhang ◽  
Yankai Lv ◽  
Tao Wang ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Disulfide Bonds ◽  
Acute Hypoxia ◽  
Extracellular Domain ◽  
Left Ventricular ◽  
Extracellular Calcium ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing

Activation of the CaSR (extracellular calcium-sensing receptor) has been recognized as a critical mediator of hypoxia-induced pulmonary hypertension. Preventive targeting of the early initiating phase as well as downstream events after CaSR activation remains unexplored. As a representative of the G protein-coupled receptor family, CaSR polymerizes on cell surface upon stimulation. Immunoblotting together with MAL-PEG technique identified a reactive oxygen species-sensitive CaSR polymerization through its extracellular domain in pulmonary artery smooth muscle cells upon exposure to acute hypoxia. Fluorescence resonance energy transfer screening employing blocking peptides determined that cycteine129/131 residues in the extracellular domain of CaSR formed intermolecular disulfide bonds to promote CaSR polymerization. The monitoring of intracellular Ca 2+ signal highlighted the pivotal role of CaSR polymerization in its activation. In contrast, the blockade of disulfide bonds formation using a peptide decreased both CaSR and hypoxia-induced mitogenic factor expression as well as other hypoxic-related genes in vitro and in vivo and attenuated pulmonary hypertension development in rats. The blocking peptide did not affect systemic arterial oxygenation in vivo but inhibited acute hypoxia-induced pulmonary vasoconstriction. Pharmacokinetic analyses revealed a more efficient lung delivery of peptide by inhaled nebulizer compared to intravenous injection. In addition, the blocking peptide did not affect systemic arterial pressure, body weight, left ventricular function, liver, or kidney function or plasma Ca 2+ level. In conclusion, a peptide blocking CaSR polymerization reduces its hypoxia-induced activation and downstream events leading to pulmonary hypertension and represents an attractive inhaled preventive alternative worthy of further development.

scholarly journals Non-Ionizing Radiation for Cardiac Human Amniotic Mesenchymal Stromal Cell Commitment: A Physical Strategy in Regenerative Medicine

2018 ◽  
Vol 19 (8) ◽  
pp. 2324 ◽  
Author(s):  
Mario Ledda ◽  
Enrico D’Emilia ◽  
Maria Lolli ◽  
Rodolfo Marchese ◽  
Claudio De Lazzari ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Therapy ◽  
Adult Stem Cells ◽  
Myocardial Damage ◽  
Stem Cell Differentiation ◽  
Differentiation Markers ◽  
Innovative Strategy ◽  
Cell Commitment

Cell therapy is an innovative strategy for tissue repair, since adult stem cells could have limited regenerative ability as in the case of myocardial damage. This leads to a local contractile dysfunction due to scar formation. For these reasons, refining strategy approaches for “in vitro” stem cell commitment, preparatory to the “in vivo” stem cell differentiation, is imperative. In this work, we isolated and characterized at molecular and cellular level, human Amniotic Mesenchymal Stromal Cells (hAMSCs) and exposed them to a physical Extremely Low Frequency Electromagnetic Field (ELF-EMF) stimulus and to a chemical Nitric Oxide treatment. Physically exposed cells showed a decrease of cell proliferation and no change in metabolic activity, cell vitality and apoptotic rate. An increase in the mRNA expression of cardiac and angiogenic differentiation markers, confirmed at the translational level, was also highlighted in exposed cells. Our data, for the first time, provide evidence that physical ELF-EMF stimulus (7 Hz, 2.5 µT), similarly to the chemical treatment, is able to trigger hAMSC cardiac commitment. More importantly, we also observed that only the physical stimulus is able to induce both types of commitments contemporarily (cardiac and angiogenic), suggesting its potential use to obtain a better regenerative response in cell-therapy protocols.

scholarly journals Putative Autocleavage of Outer Capsid Protein μ1, Allowing Release of Myristoylated Peptide μ1N during Particle Uncoating, Is Critical for Cell Entry by Reovirus

2004 ◽  
Vol 78 (16) ◽  
pp. 8732-8745 ◽  
Author(s):  
Amy L. Odegard ◽  
Kartik Chandran ◽  
Xing Zhang ◽  
John S. L. Parker ◽  
Timothy S. Baker ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Structural Changes ◽  
General Mechanism ◽  
Membrane Penetration ◽  
Outer Capsid Protein ◽  
Core Proteins ◽  
Peptide Release ◽  
Outer Capsid

ABSTRACT Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

scholarly journals The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

2020 ◽  
Author(s):  
Emma Carley ◽  
Rachel K. Stewart ◽  
Abigail Zieman ◽  
Iman Jalilian ◽  
Diane. E. King ◽  
...  
Keyword(s):  
Cell Fate ◽  
Control Cell ◽  
Nuclear Lamina ◽  
Epidermal Differentiation ◽  
Keratinocyte Differentiation ◽  
Linc Complex ◽  
Force Transmission ◽  
Cell Fate Decisions

AbstractWhile the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

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