scholarly journals Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.

1994 ◽  
Vol 125 (1) ◽  
pp. 205-214 ◽  
Author(s):  
P Rousselle ◽  
M Aumailley
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Culture Media ◽  
Human Keratinocytes ◽  
Cell Types ◽  
Heat Denaturation ◽  
Integrin Subunit ◽  
Cellular Interactions ◽  
Primary Human Keratinocytes ◽  
Established Cell

Kalinin was purified from squamous cell carcinoma (SCC25) spent culture media using an immunoaffinity column prepared from the mAb BM165. The affinity-purified material was separated by SDS-PAGE into three bands of 165-155, 140, and 105 kD identical to those obtained from normal human keratinocyte cultures and previously identified as kalinin. Kalinin promoted adhesion of a large number of normal cells and established cell lines with an activity similar to other adhesion molecules such as the laminin-nidogen complex, fibronectin, or collagen IV. However, kalinin was a much better substrate than laminin-nidogen complex for adhesion of cells of epithelial origin including primary human keratinocytes. Adhesion to kalinin was followed by cell shape changes ranging from rounded to fully spread cells depending on the cell types. The adhesion-promoting activity of kalinin was conformation dependent and was abolished by heat denaturation. mAb BM165 prevented cell adhesion to kalinin but not to other extracellular matrix substrates. However, either complete or partial inhibition was observed with different cells suggesting the existence of at least two cell-binding sites on the kalinin molecule. Experiments inhibiting cell adhesion with function-blocking anti-integrin subunit antibodies indicated that both alpha 3 beta 1 and alpha 6 beta 1 integrins are involved in the cellular interactions with kalinin, while for cell adhesion to classical mouse Engelbreth-Holm-Swarm laminin only alpha 6 beta 1 integrins, and not alpha 3 beta 1, appeared to be functional. Altogether, these results suggest that kalinin may fulfill additional functions than laminin, particularly for epithelial cells.

scholarly journals Lycopene Modulates THP1 and Caco2 Cells Inflammatory State through Transcriptional and Nontranscriptional Processes

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Njock Makon-Sébastien ◽  
Fouchier Francis ◽  
Seree Eric ◽  
Villard Pierre Henri ◽  
Landrier Jean François ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Culture Media ◽  
Cell Types ◽  
Ros Production ◽  
M Cell ◽  
Low Concentration ◽  
Inflammatory State ◽  
Proinflammatory Gene ◽  
Inflammatory Effect

We revisited the action of a carotenoid, the lycopene, on the expression of proinflammatory genes, reactive oxygen species (ROS) production, and metalloprotease (MMP9) activity. THP1 and Caco2 cell lines were used asin vitromodels for the two main cell types found in intestine tissue, that is, monocytes and epithelial cells. Proinflammatory condition was induced using either phorbol ester acetate (PMA), lipopolysaccharide (LPS) or tumor necrosis factor (TNF). In THP1 cells, short term pretreatment (2 h) with a low concentration (2 μM) of lycopene reinforce proinflammatory gene expression. The extent of the effect of lycopene is dependent on the proinflammtory stimulus (PMA, LPS or TNF) used. Lycopene enhanced MMP9 secretion via a c-AMP-dependent process, and reduced ROS production at higher concentrations than 2 μM. Cell culture media, conditioned by PMA-treated monocytes and then transferred on CaCo-2 epithelial cells, induced a proinflammatory state in these cells. The extent of this inflammatory effect was reduced when cells has been pretreated (12 h) with lycopene. At low concentration (2 μM or less), lycopene appeared to promote an inflammatory state not correlated with ROS modulation. At higher concentration (5 μM–20 μM), an anti-inflammatory effect takes place as a decrease of ROS production was detected. So, both concentration and time have to be considered in order to define the exact issue of the effect of carotenoids present in meals.

scholarly journals Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit.

1991 ◽  
Vol 115 (4) ◽  
pp. 1137-1148 ◽  
Author(s):  
A P Skubitz ◽  
P C Letourneau ◽  
E Wayner ◽  
L T Furcht
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Neurite Outgrowth ◽  
Cell Types ◽  
Integrin Subunit ◽  
Globular Domain ◽  
Heparin Binding ◽  
Beta 1 Integrin ◽  
A Chain ◽  
Carboxy Terminal

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.

Amino- and Carboxy-functionalized Nano- and Microstructured Surfaces for Evaluating the Impact of Non-biological Stimuli on Adhesion, Proliferation and Differentiation of Primary Skin-Cells

2009 ◽  
Vol 1187 ◽  
Author(s):  
Petra J. Kluger ◽  
Marc Panas ◽  
Lena Schober ◽  
Guenter E. M. Tovar ◽  
Heike Mertsching ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Human Keratinocytes ◽  
Cell Types ◽  
Primary Human Keratinocytes ◽  
Chemical Functionalization ◽  
Skin Cells ◽  
Functionalized Surfaces ◽  
Microstructured Surfaces ◽  
Primary Skin Cells ◽  
The Impact

AbstractTo gain basic insight into the impact of non-biological features on cells’ behaviour, primary skin-cells, keratinocytes and fibroblasts, were cultured on amine-functionalized or carboxy-functionalized planar, nano- or microstructured surfaces. Sintered layers of silica nano- or microparticles were used to fabricate structures in the range of naturally occurring structure-sizes. Organo-chemical functionalization was achieved using organo-functional silanes. Primary human keratinocytes and fibroblasts were isolated from human foreskin and cultivated on the modified interfaces. Both cell-types displayed specific proliferation behaviour, depending on surface topography and chemical functionalization: Keratinocytes showed significantly better proliferation on amino-functionalized surfaces than on carboxy-functionalized surfaces. On amino-functional surfaces decree-topography. Fibroblasts, in contrast, tended to proliferate stronger on carboxylated surfaces. Immunohistological staining proofed that actin and vinculin, which is involved in the formation of focal adhesions, were expressed on all modified surfaces, thus revealing intact cytoskeleton and cell-substrate contacts.

scholarly journals Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.

1990 ◽  
Vol 110 (6) ◽  
pp. 2145-2155 ◽  
Author(s):  
A Sonnenberg ◽  
C J Linders ◽  
P W Modderman ◽  
C H Damsky ◽  
M Aumailley ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Types ◽  
Integrin Subunit ◽  
Integrin Receptors ◽  
Cell Binding ◽  
Vitronectin Receptor ◽  
Beta 1 Integrin ◽  
Integrin Alpha 6 ◽  
Alpha V Beta 3 ◽  
Integrin Family

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.

scholarly journals Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation

2003 ◽  
Vol 23 (3) ◽  
pp. 777-790 ◽  
Author(s):  
Martin Leverkus ◽  
Martin R. Sprick ◽  
Tina Wachter ◽  
Thilo Mengling ◽  
Bernd Baumann ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Proteasome Inhibitors ◽  
Human Keratinocytes ◽  
Cell Types ◽  
Primary Cells ◽  
Primary Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Effector Caspase ◽  
Apoptosis Protein ◽  
Induced Apoptosis

ABSTRACT Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-κB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-κB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

scholarly journals Cell-cell signaling elicits local Ca2+ transients in melanocyte dendrites and dendritic spine-like structures

2018 ◽  
Author(s):  
Rachel L. Belote ◽  
Sanford M. Simon
Keyword(s):  
Dendritic Spines ◽  
Cell Communication ◽  
Human Keratinocytes ◽  
Cell Types ◽  
Primary Human Keratinocytes ◽  
Human Melanocytes ◽  
Neuronal Functions ◽  
Dendritic Branches ◽  
Cell Cell

AbstractCompartmentalized cytoplasmic fluctuations of Ca2+ within dendrites and dendritic spines regulate a variety of neuronal functions. Like some neurons and glia, melanocytes are neural crest derived and possess dendrites (Adameyko et al., 2009; Erickson et al., 1992; Fitzpatrick and Szabo, 1959). Here, we show that primary human melanocytes, when observed in situ have extensive dendritic branches with dendritic spines similar to neurons. When co-cultured with primary human keratinocytes, they have local Ca2+ transients within these spines and within the dendrites. These are elicited by secreted factors from adjacent keratinocytes. Thus other cell types with dendrites are capable of compartmentalized Ca2+ fluctuations in response to cell-cell communication. Furthermore, our observations within intact human skin suggest a more complex communication network between adjacent melanocytes and keratinocytes, and thus a more complex physiology to skin than previous appreciated.

Identification of receptors binding fibronectin and laminin on fetal rat lung cells

1996 ◽  
Vol 270 (3) ◽  
pp. L459-L468 ◽  
Author(s):  
I. Caniggia ◽  
J. Liu ◽  
R. Han ◽  
J. Wang ◽  
A. K. Tanswell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fetal Lung ◽  
Cell Types ◽  
Lung Epithelial Cells ◽  
Integrin Subunit ◽  
Fibronectin Receptor ◽  
Lung Epithelial ◽  
Integrin Binding Site ◽  
Beta1 Integrin ◽  
A Chain

Fibronectin and laminin have been implicated in regulating lung morphogenesis. In the present study, the cell surface receptors involved in fetal lung cell binding to laminin and fibronectin were identified. Messages for alpha5- and beta1-integrin subunits were detected in both fetal lung epithelial cells and fibroblasts. The presence of alpha5 beta1 -integrin on both cell types was demonstrated by immunocytochemistry and confirmed by cell adhesion experiments with fibronectin and RGD-containing peptides. Epithelial cells adhered more readily to laminin than fibroblasts. The alpha4 beta1-integrin, and RGD-independent fibronectin receptor, was weakly expressed on either cell type. Both cell types expressed alpha6-integrin subunit mRNA and stained immunopositive for the alpha6-subunit. Although either cell type expressed nonintegrin 67-kDa laminin-elastin receptor mRNA, no positive immunoreactivity for this laminin-elastin binding protein was detected. None of these findings explain the enhanced attachment of distal fetal lung epithelial cells to laminin compared with fibroblasts. Previously, we have reported that epithelial cells were enriched in alpha3-integrin subunit mRNA and protein expression. Herein, we found that epithelial cell attachment to laminin was nearly completely inhibited by alpha3- but only partially by alpha6 -monoclonal antibodies. A peptide near the globular region at the long arm of the laminin A-chain, which contained the IKVAV sequence, and the laminin A-chain amino acid sequence representing the alpha3 beta1 -integrin binding site, inhibited the adherence of epithelial cells to laminin. Fetal lung epithelial cells attached to substrata coated with the alpha3 beta1-integrin binding site peptide and the peptide containing the IKVAV sequence. These data suggest that both fetal lung cell types bind to fibronectin via the fibronectin receptor, alpha5 beta1, and fetal lung epithelial cells interact with laminin via alpha3 beta1 and proteins that recognize the IKVAV-containing sequence on the laminin A-chain.

scholarly journals Coupled expression and colocalization of 140K cell adhesion molecules, fibronectin, and laminin during morphogenesis and cytodifferentiation of chick lung cells.

1986 ◽  
Vol 103 (3) ◽  
pp. 1073-1090 ◽  
Author(s):  
W T Chen ◽  
J M Chen ◽  
S C Mueller
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Cell Adhesion ◽  
Epithelial Cells ◽  
Smooth Muscle Cells ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Cell Types ◽  
Lung Cells ◽  
Embryonic Lung

We have analyzed the expression and distribution of fibronectin, laminin, and the 140K cell adhesion molecules (140K complex) in embryonic chick lung cells by a combination of biochemical and immunofluorescent approaches. The 140K complex was identified by monoclonal antibody JG22E as a complex of glycoproteins averaging 140,000 Mr and has been implicated in vitro as a receptor for fibronectin and laminin. Our studies provide the first description that the 140K complex is developmentally regulated, and that the 140K complex appears to be involved in adhesion of epithelial and endothelial cells during morphogenesis. We have shown that the 140K complex is expressed in high quantity in embryonic lung cell types, but is markedly reduced in all of the differentiated cell types except smooth muscle. Embryonic lung cells are enriched in 140K complex on portions of cells in close proximity to areas rich in fibronectin. For example, during the formation of airways and alveolar tissues, 140K complex is concentrated at the basal surfaces of epithelial cells adjacent to fibronectin. Likewise, during the angiogenic invasion of capillaries into lung mesenchyme, the 140K complex becomes localized at sites on the basal surfaces of endothelial cells in close contact with fibronectin. Finally, cytodifferentiating lung smooth muscle cells show unusually high levels of 140K complex, fibronectin, and laminin that persist into the adult. In contrast to fibronectin, laminin is found to be uniformly distributed in the basement membranes of differentiating epithelial cells. It becomes prominent in adult alveolar epithelium and airway epithelium concomitant with a reduction or loss of 140K complex and fibronectin at cell-basement membrane attachment sites. Surprisingly, laminin is also present in a punctate pattern in the mesenchyme of early lung buds, however, laminin, fibronectin, and 140K complex are greatly reduced or lost during mesenchymal maturation. Our results are consistent with the active participation of the 140K complex in cell-to-matrix adhesion during morphogenesis of alveolar walls and cytodifferentiation of mesenchymal and smooth muscle cells.

Three-Dimensional Endothelium-Lined Microfabricated Channels as in Vitro assays for Sickle Cell Vaso-Occlusion

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4790-4790
Author(s):  
Michelle Tsai ◽  
Thomas P. Hunt ◽  
Daniel A. Fletcher ◽  
Wilbur Aaron Lam
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell ◽  
Blood Cells ◽  
Cell Types ◽  
Cellular Interactions ◽  
Cell Deformability ◽  
Soluble Factors ◽  
Different Cell Types

Abstract Vaso-occlusion in sickle cell disease is fundamentally biophysical in nature, involving a complex set of cellular interactions. Originally attributed solely to the entrapment of abnormally rigid sickle red cells (RBCs) in the microcirculation, this process is now known to involve the decreased deformability of white blood cells (WBCs) and increased endothelial adhesion to different cell types (sickle RBCs, reticulocytes, WBCs, platelets). These biophysical interactions, which are also mediated biochemically by a variety of soluble factors (coagulation proteins, inflammatory mediators, reactive oxygen species, free hemoglobin, etc.), then ultimately lead to microvascular obstruction. However, in vitro experimental approaches that measure the vaso-occlusive properties of sickle blood cells have been unable to separate the contributions of decreased cell deformability and cell-cell adhesion in a single assay. Historically, cell deformability is measured using techniques such as micropipette aspiration, micropore filtration, and ektacytometry, whereas adhesive interactions between the endothelium, blood cells, and soluble factors are assessed using endothelial-lined flow chamber assays. No existing technique effectively evaluates both cell deformability and cell adhesion simultaneously, which is required to comprehensively study sickle cell vaso-occlusion. In the current study, we present an “endothelialized” microfluidic system that simultaneously integrates cell deformability and cell adhesion under physiologic microvascular flow conditions to investigate the underlying biophysical mechanisms of sickle cell vaso-occlusion. Briefly, a layer of human umbilical vein endothelial cells (HUVECs) was cultured along the inner walls of microfabricated microchannels made of the biocompatible and optically transparent polymer polydimethylsiloxane, or PDMS (Figure). Standard lab-on-chip photolithography techniques were utilized to design and create the microchannels, which geometrically emulate a branching microvasculature network with the smallest lumen size approximately 15 μm in diameter. Then, the inner lining of the microchannels were coated with fibronectin and seeded with HUVECs, which grew to a confluent monolayer within 4–5 days and encompassed the entire inner surface in all three dimensions. Whole blood or cell suspensions can then be perfused into the system, and cells can be visualized under flow using standard immunofluorescence microscopy techniques, which allows for identification of specific cellular subpopulations. Pressure and flow rate can be tightly controlled, and cell transit time, time to cell aggregation or obstruction of the system can be recorded using automated image processing software. The effect of different cell types and biologic modifiers (i.e. cytokines, coagulation factors, etc) on in vitro vaso-occlusion can be systematically analyzed and quantified. Furthermore, adhesion-blocking antibodies and drugs that increase cell deformability can be used to quantify the vaso-occlusive effect of adhesion versus cell rigidity. As this system physically constrains cells three-dimensionally in lumens the size of the human microvasculature, physical cellular interactions that are likely to occur in vivo and the endothelial cell response to these processes can also be directly evaluated with live cell fluorescence, immunofluorescence or western blotting. Overall, this system will provide a quantitative and controlled approach to investigate the underlying complex biophysical processes that govern sickle cell vaso-occlusion. In addition, this platform will also serve as a useful complimentary technology to in vivo experiments using sickle cell mouse models. Figure Figure

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