scholarly journals Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit.

1993 ◽  
Vol 121 (1) ◽  
pp. 171-178 ◽  
Author(s):  
F Balzac ◽  
A M Belkin ◽  
V E Koteliansky ◽  
Y V Balabanov ◽  
F Altruda ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Subcellular Localization ◽  
Functional Properties ◽  
Cultured Cells ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Variant Form ◽  
Integrin Subunit ◽  
Stable Transfectants

We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties.

scholarly journals Proteolytic processing of endogenous and recombinant beta 4 integrin subunit.

1992 ◽  
Vol 118 (4) ◽  
pp. 951-959 ◽  
Author(s):  
F G Giancotti ◽  
M A Stepp ◽  
S Suzuki ◽  
E Engvall ◽  
E Ruoslahti
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Cytoplasmic Domain ◽  
Basement Membranes ◽  
Proteolytic Processing ◽  
In Vitro Assays ◽  
Integrin Subunit ◽  
Calcium Dependent

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.

Activation of human keratinocyte migration on type I collagen and fibronectin

1990 ◽  
Vol 96 (2) ◽  
pp. 197-205
Author(s):  
M. Guo ◽  
K. Toda ◽  
F. Grinnell
Keyword(s):  
Type I Collagen ◽  
Cultured Cells ◽  
Focal Adhesions ◽  
Human Keratinocytes ◽  
Type I ◽  
Integrin Subunit ◽  
Basal Cells ◽  
Keratinocyte Migration ◽  
Beta 1 Integrin ◽  
Cells Cultured

The purpose of our studies was to learn more about the regulation of keratinocyte migration. Human keratinocytes freshly harvested from skin were relatively immotile cells, whereas keratinocytes harvested from cell culture migrated on type I collagen or fibronectin as measured in a phagokinesis assay. Development of migratory competence by keratinocytes varied depending on the culture substratum. Cells cultured on plastic were activated more quickly and to a greater extent than cells cultured on dermis. The effect of the culture substratum on migratory competence was reversible. That is, cells cultured on plastic showed reduced activity after subculture on dermis. Cells cultured on dermis showed increased activity after subculture on plastic. Freshly isolated as well as cultured keratinocytes contained beta 1 integrin subunits, but only cultured cells were able to organize the subunits into focal adhesions. These adhesion sites also contained vinculin. In epidermal explants, beta 1 integrin subunits were mostly in basal cells, often more prominent between lateral cell borders than at the epidermal-dermal interface. In keratinocytes that migrated out of skin explants, there appeared to be an increase in the intensity of beta 1 integrin subunit immunostaining, possibly because of the change in shape of migrating cells. Also, beta 1 integrin subunits were found around and beneath migrating keratinocytes. These results show that changes in the distribution of beta 1 integrin subunits accompany development of migratory competence.

scholarly journals Parallel Isolation and Characterization of Porcine Smooth Muscle, Endothelial and Mesenchymal Stromal Cells for Bioengineering Applications

2021 ◽  
Author(s):  
Sara Morini ◽  
Iris Pla-Palacín ◽  
Pilar Sainz-Arnal ◽  
Natalia Sánchez-Romero ◽  
Maria Falceto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cultured Cells ◽  
Umbilical Vein ◽  
Building Blocks ◽  
Cell Types ◽  
Aortic Smooth Muscle ◽  
Isolation And Characterization ◽  
Umbilical Vein Endothelial Cells

Abstract There is significant interest in the pig as the animal model of choice for organ transplantation and the study of tissue engineering (TE) products and applications. Currently, efforts are being taken to bioengineer solid organs to reduce donor shortages for transplantation. For complex organs such as the lung, heart, and liver, the vasculature represents a fundamental feature. Thus, to generate organs with a functional vascular network, the different cells constituting the building blocks of the blood vessels should be procured. However, due to species' specificities, porcine cell isolation, expansion, and characterization are not entirely straightforward compared to human cell procurement. Here, we report the establishment of simple and suitable methods for the isolation and characterization of distinct porcine cells for bioengineering purposes.We successfully isolated, expanded and characterized porcine bone marrow-derived mesenchymal stromal (pBM-MSC), aortic smooth muscle (pASMC), and umbilical vein endothelial cells (pUVEC). We demonstrated that the three cell types showed specific immunophenotypical features. Moreover, we demonstrated that pBM-MSC could preserve their multipotency in vitro, and pUVEC were capable of maintaining their functionality in vitro.These cultured cells could be further expanded and represent a useful cellular tool for TE purposes (i.e., for recellularization approaches of vascularized organs or in vitro angiogenesis studies).

243. In vitro culture significantly reduces differences in gene expression profiles between myometrial and fibroid smooth muscle cells

2005 ◽  
Vol 17 (9) ◽  
pp. 96
Author(s):  
M. Zaitseva ◽  
P. A. W. Rogers
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Muscle Cells ◽  
Compare Gene Expression

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMC) have been widely used as a model for the study of fibroid growth. Although it has been shown that FSMC can behave differently in culture to MSMC, it is not clear how relevant the cultured cells and their responses are to the in-vivo situation. The aim of the present study was to compare gene expression profiles of M and F tissue to cells isolated from the same tissue and cultured for up to 3 passages. M and F were collected from hysterectomy specimens (n = 6), part was snap frozen for RNA and the rest used to isolate SMC, which were cultured for 3 passages and RNA was collected at passage 0 (P0) and 3 (P3). 36 microarrays were performed on 8K human cDNA slides, 6 per each specimen (3 for M and 3 for F: tissue, cell at P0 and P3) against reference RNA. Analysis revealed significant differences between tissues and cultured cells. Independent clustering assigned tissues versus cells into two distinct groups based on their expression profiles. Parametric ANOVA with Benjamini-Hochberg correction and post-hoc testing was used to determine similarities and differences between tissues and cells. 128 genes were found to be statistically different between M and F tissue, 66 between MSMC and FSMC at P0, and only 9 at P3. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Similar numbers of genes were up regulated as were down regulated. Expression profiles of genes of interest including estrogen receptor α and progesterone receptor were also validated using real-time PCR. This is the first study to compare gene expression of in vivo and in vitro fibroid and myometrial SMC. The results demonstrate that large changes occur in SMC gene expression in culture, reducing differences between myometrial and fibroid cells. This study indicates that results of in vitro studies should be interpreted with caution as many genes have an altered gene expression profile in culture.

Functions of the cytoplasmic domain of the beta PS integrin subunit during Drosophila development

Development ◽  
1994 ◽  
Vol 120 (1) ◽  
pp. 91-102 ◽  
Author(s):  
Y. Grinblat ◽  
S. Zusman ◽  
G. Yee ◽  
R.O. Hynes ◽  
F.C. Kafatos
Keyword(s):  
Germ Line ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
P Element ◽  
Integrin Subunit ◽  
Wild Type ◽  
Mutant Proteins ◽  
Adhesive Interactions

Integrins constitute a family of membrane-spanning, heterodimeric proteins that mediate adhesive interactions between cells and surrounding extracellular matrices (or other cells) and participate in signal transduction. We are interested in assessing integrin functions in the context of developing Drosophila melanogaster. This report, using mutants of the beta PS subunit encoded by the myospheroid (mys) locus, analyzes the relationships between integrin protein structure and developmental functions in an intact organism. As a first step in this analysis, we demonstrated the ability of a fragment of wild-type mys genomic DNA, introduced into the germ line in a P-element vector P[mys+], to rescue phenotypes attributed to lack of (or defects in) the endogenous beta PS during several discrete morphogenetic events. We then produced in vitro a series of modifications of the wild-type P[mys+] transposon, which encode beta PS derivatives with mutations within the small and highly conserved cytoplasmic domain. In vivo analysis of these mutant transposons led to the following conclusions. (1) The cytoplasmic tail of beta PS is essential for all developmental functions of the protein that were assayed. (2) An intron at a conserved position in the DNA sequence encoding the cytoplasmic tail is thought to participate in important alternative splicing events in vertebrate beta integrin subunit genes, but is not required for the developmental functions of the mys gene assayed here. (3) Phosphorylation on two conserved tyrosines found in the C terminus of the beta PS cytoplasmic tail is not necessary for the tested developmental functions. (4) Four highly conserved amino acid residues found in the N-terminal portion of the cytoplasmic tail are important but not critical for the developmental functions of beta PS; furthermore, the efficiencies with which these mutant proteins function during different morphogenetic processes vary greatly, strongly suggesting that the cytoplasmic interactions involving PS integrins are developmentally modulated.

scholarly journals Syndecan-4 regulates localization, activity and stability of protein kinase C-alpha

2004 ◽  
Vol 378 (3) ◽  
pp. 1007-1014 ◽  
Author(s):  
Eunyoung KEUM ◽  
Yeonhee KIM ◽  
Jungyean KIM ◽  
Soojin KWON ◽  
Yangmi LIM ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Heparan Sulphate ◽  
Critical Role ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Prolonged Treatment ◽  
Kinase C

During cell–matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibres. We have shown previously that the syndecan-4 cytoplasmic domain directly binds to and activates PKC-α (protein kinase C-α) in vitro [Oh, Woods and Couchman (1997) J. Biol. Chem. 272, 8133–8136]. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, we showed that syndecan-4 interacted with PKC-α in vivo and that this interaction was mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increased the extent of interaction between syndecan-4 and PKC-α. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increased the level of endogenous PKC-α and enhanced the translocation of PKC-α into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibited a slowed down-regulation of PKC-α in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-α immunocomplex kinase assays also showed that syndecan-4 overexpression increased the activity of membrane PKC-α. Taken together, these results suggest that syndecan-4 interacts with PKC-α in vivo and regulates its localization, activity and stability.

α1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins

2001 ◽  
Vol 356 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Klemens LÖSTER ◽  
Dörte VOSSMEYER ◽  
Werner HOFMANN ◽  
Werner REUTTER ◽  
Kerstin DANKER
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion ◽  
Protein Interactions ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Adhesion Receptors ◽  
Focal Adhesion Formation

Integrins are heterodimeric adhesion receptors consisting of α- and β-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein–protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic α1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the α1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on α1β1-specific substrates. The α1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, α-actinin and pp125FAK. It seems to account for α1-specific signalling, since when peptides that mimic the cytoplasmic domain of α1 are transferred into cells, they influence α1β1-specific adhesion, presumably by competing for binding partners. For α1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

The Cytoplasmic Domain of α6A Integrin Subunit Is an in Vitro Substrate for Protein Kinase C

1995 ◽  
Vol 216 (1) ◽  
pp. 232-235 ◽  
Author(s):  
Clotilde Gimond ◽  
Annemieke de Melker ◽  
Monique Aumailley ◽  
Arnoud Sonnenberg
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Kinase C

scholarly journals Aging impairs smooth muscle-mediated regulation of aortic stiffness: a defect in shock absorption function?

2014 ◽  
Vol 307 (8) ◽  
pp. H1252-H1261 ◽  
Author(s):  
Yuan Z. Gao ◽  
Robert J. Saphirstein ◽  
Rina Yamin ◽  
Bela Suki ◽  
Kathleen G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Aortic Stiffness ◽  
Kinase Inhibitor ◽  
Additive Model ◽  
Focal Adhesions ◽  
Shock Absorption ◽  
Absorption Function ◽  
Material Stiffness ◽  
Old Mice

Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of NG-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90–200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aorta's normal shock absorption function in response to stressors.

scholarly journals Bioprintable Lung Extracellular Matrix Hydrogel Scaffolds for 3D Culture of Mesenchymal Stromal Cells

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2350
Author(s):  
Bryan Falcones ◽  
Héctor Sanz-Fraile ◽  
Esther Marhuenda ◽  
Irene Mendizábal ◽  
Ignacio Cabrera-Aguilera ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
3D Culture ◽  
Focal Adhesions ◽  
Fold Increase ◽  
Paracrine Factors ◽  
Hydrogel Scaffolds ◽  
Vitro Model ◽  
Cells Cultured

Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell–matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.

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