scholarly journals Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover.

1994 ◽  
Vol 5 (12) ◽  
pp. 1281-1288 ◽  
Author(s):  
D J Mooney ◽  
L K Hansen ◽  
R Langer ◽  
J P Vacanti ◽  
D E Ingber
Keyword(s):  
Extracellular Matrix ◽  
Cell Shape ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Rat Hepatocytes ◽  
Defined Medium ◽  
Type I ◽  
Mrna Levels ◽  
Mechanical Loads ◽  
In Cells

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.

scholarly journals Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum

1990 ◽  
Vol 268 (1) ◽  
pp. 225-230 ◽  
Author(s):  
A E Canfield ◽  
R P Boot-Handford ◽  
A M Schor
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Shape ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Gels ◽  
Cells Cultured

Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a three-dimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steady-state mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin- or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

scholarly journals Morphodynamics facilitate cancer cells to navigate 3D extracellular matrix

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Christopher Z. Eddy ◽  
Helena Raposo ◽  
Aayushi Manchanda ◽  
Ryan Wong ◽  
Fuxin Li ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Motility ◽  
Cancer Cells ◽  
Cell Shape ◽  
Type I Collagen ◽  
Cell Function ◽  
Type I ◽  
Step Size ◽  
Mesoscale Dynamics ◽  
Hidden Markov Process

AbstractCell shape is linked to cell function. The significance of cell morphodynamics, namely the temporal fluctuation of cell shape, is much less understood. Here we study the morphodynamics of MDA-MB-231 cells in type I collagen extracellular matrix (ECM). We systematically vary ECM physical properties by tuning collagen concentrations, alignment, and gelation temperatures. We find that morphodynamics of 3D migrating cells are externally controlled by ECM mechanics and internally modulated by Rho/ROCK-signaling. We employ machine learning to classify cell shape into four different morphological phenotypes, each corresponding to a distinct migration mode. As a result, we map cell morphodynamics at mesoscale into the temporal evolution of morphological phenotypes. We characterize the mesoscale dynamics including occurrence probability, dwell time and transition matrix at varying ECM conditions, which demonstrate the complex phenotype landscape and optimal pathways for phenotype transitions. In light of the mesoscale dynamics, we show that 3D cancer cell motility is a hidden Markov process whereby the step size distributions of cell migration are coupled with simultaneous cell morphodynamics. Morphological phenotype transitions also facilitate cancer cells to navigate non-uniform ECM such as traversing the interface between matrices of two distinct microstructures. In conclusion, we demonstrate that 3D migrating cancer cells exhibit rich morphodynamics that is controlled by ECM mechanics, Rho/ROCK-signaling, and regulate cell motility. Our results pave the way to the functional understanding and mechanical programming of cell morphodynamics as a route to predict and control 3D cell motility.

Phenotypic Changes of Rabbit Mandibular Condylar Cartilage Cells in Culture

1990 ◽  
Vol 69 (11) ◽  
pp. 1753-1758 ◽  
Author(s):  
F.E. Engel ◽  
A.G. Khare ◽  
B.D. Boyan
Keyword(s):  
Type I Collagen ◽  
Antibody Activity ◽  
Type I ◽  
Mrna Levels ◽  
Condylar Cartilage ◽  
Phenotypic Changes ◽  
Time Points ◽  
Mandibular Condylar Cartilage ◽  
In Cells

The present study describes the behavior of mandibular condylar cartilage (MCC) cells as a function of time in primary culture, since it is not yet clear whether these cells maintain their phenotype in culture. MCC cells from New Zealand white rabbits were seeded at high density and cultured in DMEM containing 50 μg/mL ascorbic acid and 10% fetal bovine serum. These cells appeared as a heterogeneous population and changed their shape, size, and refractivity as cultures aged. Cartilage-like cells, which always dominated the culture, were infiltrated with a minority of fibroblast-like cells. Cell number increased progressively, and cultures reached confluence at nine days. Antibody activity for cartilage-specific glycosaminoglycan was determined by ELISA assay. This reaction reached a maximum at six days and decreased thereafter. Cultures stained with Alcian blue (pH 1.0) supported these results. Cytoplasmic mRNA analysis indicated that the transcription of type II collagen gene was present at all time points. Type I collagen and alkaline phosphatase mRNA levels showed progressive increases from 12 h to nine days, with significantly higher values in cells cultured for six, nine, and 12 days than in cells collected from earlier time points. These results suggest that in our present culture system, MCC cells undergo phenotypic changes that resemble their maturation processes in vivo.

scholarly journals Mechanical strain increases type I collagen expression in pulmonary fibroblasts in vitro

2000 ◽  
Vol 88 (1) ◽  
pp. 203-209 ◽  
Author(s):  
Ellen C. Breen
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Mechanical Strain ◽  
Type I ◽  
Mrna Levels ◽  
Pulmonary Fibroblasts ◽  
Matrix Interactions ◽  
Proline Incorporation ◽  
Extracellular Matrix Interactions

Tissue remodeling is an adaptive response to mechanical tension in the lung. However, the role of pulmonary fibroblasts in this response has not been well characterized. This study investigates the influence of extracellular matrix on the response of fibroblasts to mechanical strain. Cells were cultured on flexible-bottom surfaces coated with fibronectin, laminin, or elastin and exposed to strain. Under these conditions, fibroblasts align perpendicular to the force vector. This stimulus results in an increase in α1(I) procollagen mRNA in cells cultured on laminin or elastin but not fibronectin. Increased α1(I) procollagen mRNA was detected 6 h after exposure to strain and reached control levels by 72 h. [3H]proline incorporation into newly synthesized procollagen reflects changes in mRNA levels. Strained fibroblasts cultured on laminin or elastin incorporated 190 and 114%, respectively, more [3H]proline into procollagen than did unstrained cells. No difference was detected in strained fibroblasts cultured on fibronectin. These results suggest that fibroblasts respond to mechanical strain in vitro , and this response is signaled by cell-extracellular matrix interactions.

Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

1995 ◽  
Vol 108 (6) ◽  
pp. 2311-2320 ◽  
Author(s):  
D.J. Mooney ◽  
R. Langer ◽  
D.E. Ingber
Keyword(s):  
Extracellular Matrix ◽  
Cell Shape ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Cytochalasin D ◽  
Short Exposure ◽  
Type I ◽  
Cell Extension ◽  
Actin Microfilament ◽  
The Matrix

This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell ‘shape’ changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals THE FEATURES OF THE FACE SKIN CONSTRUCTION THAT INFLUENCE ON THE FORMATION OF CICATRICAL TISSUES DURING SUGICAL INTERVENTIONS

2019 ◽  
pp. 19-23
Author(s):  
K.P. Lokes ◽  
D.S. Avetikov ◽  
S.O. Stavitsky ◽  
O.O. Rozkolupa ◽  
N.S. Lutsenko
Keyword(s):  
Extracellular Matrix ◽  
Head And Neck ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Rapid Development ◽  
Maxillofacial Surgery ◽  
Type Iii Collagen ◽  
Type I ◽  
Amino Terminal ◽  
Functional Features

Formation of pathological scars of maxillofacial localization after surgery is a significant and widespread problem of modern surgical stomatology and maxillofacial surgery. A significant percentage of patients who needs planned and urgent surgical interventions cause rapid development of reconstructive-restorative surgery of the maxillofacial region. The analysis of domestic and foreign literary sources was devoted to the peculiarities of the structure of the skin of the head and neck and the optimization of the skin incisions of this localization. Functional features of human skin depend on the mechanical properties of the dermis, which provides elasticity and resistance to stretching. Changes in the biomechanics of the dermis occur during aging, excessive insolation, scarring, and fibrosis. In addition, mechanical changes in the extracellular matrix of the skin affect the activity and phenotype of the fibroblasts, which adapt the stiffness of the cytoskeleton. Extracellular matrix stiffness defines and maintains cell identity and influences the proliferation, differentiation, migration and expression of skin cells. The extracellular matrix has been regarded for a long time as a structure with simple architectonics. But, due to modern research, it is known that this complex formation is highly specialized. The different classes of macromolecules that make up the extracellular matrix determine its biological functions. For example, collagen proteins are responsible for the tensile strength of tissues, proteoglycans and glycosaminoglycan are important for hydration and compression resistance, and glycoproteins such as laminas facilitate cell attachment. The largest structures of the extracellular matrix are elastin fibers, which are mainly localized in tissues subject to high mechanical stress, such as skin, lungs, or arteries. These structures represent a very complex organization whose core consists of elastin surrounded by a mantle of microfibrils. Collagen proteins in the dermis contain mainly type I collagen (85% - 90%) with smaller amounts of type III collagen (10% - 15%). Skin fibroblasts synthesize individual collagen type I and III polypeptide chains as precursor molecules, called procollagens. During the formation of insoluble collagen fibrils, specific proteases break down the carboxy- and amino-terminal domains, forming pN-collagen (procollagen from which the carboxy-terminal domain propeptide is cleaved) and pC-collagen (procollagen, from which the amino-terminal propeptide is cleaved). Because type I and III procollagen, pN-collagen and pC-collagen are precursors of mature collagen molecules, their level usually reflects the level of collagen biosynthesis. Collagens and elastin contain highly abundant fibrils, each of which is repeated in a sequence enriched in the conformation of polyproline II, cross-linked, insoluble in assembly and resistant to the most photolytic enzymes. The monomeric block of type I collagen consists of two extended chains α1 and one chain α2, twisted together into a triple helix. The direction of collagen and elastin fibers, according to biomechanical studies, has a significant effect on the enlargement of the wound on the head skin and the tension when closing its edges. The overwhelming reduction of tension and accordingly the improvement of reparative processes in the skin occur when the incision lines correspond to the so-called "golden spiral". Conclusion. Thus, the analysis of domestic and foreign literature sources indicates the relevance of the selected topics, the need for further studies on the biomechanical and histological substantiation of incisions, which are due to the peculiarities of the structure of the skin in the head and neck to create optimal conditions for reparative regeneration.

A type I collagen substrate increases PTH/PTHrP receptor mRNA expression and suppresses PTHrP mRNA expression in UMR106–06 osteoblast-like cells

1996 ◽  
Vol 150 (2) ◽  
pp. 299-308 ◽  
Author(s):  
S Celic ◽  
P J Chilco ◽  
J D Zajac ◽  
T J Martin ◽  
D M Findlay
Keyword(s):  
Retinoic Acid ◽  
Mrna Expression ◽  
Mrna Stability ◽  
Type I Collagen ◽  
Type I ◽  
Mrna Levels ◽  
Receptor Mrna ◽  
Pthrp Receptor ◽  
Camp Levels ◽  
In Cells

Abstract We have previously shown that the response of osteoblasts to parathyroid hormone (PTH) can be influenced at the receptor level by growth on the physiological substrate, type I collagen, or by treatment with retinoic acid. We have also shown differential expression of genes when cells of the osteoblast lineage are grown on type I collagen. The aim of this study was therefore to examine the effect of retinoic acid and growth on type I collagen on PTH/PTH-related protein (PTHrP) receptor mRNA expression in the osteosarcoma osteoblast-like cell line UMR106–06. PTH/PTHrP receptor mRNA levels, as assessed by Northern blot, of cells grown on collagen were increased up to 2-fold compared with cells on plastic and in a concentration-dependent manner with respect to collagen. An increase was seen as early as 6 h and was maintained over a 24 h period. This was not due to increased mRNA stability. Retinoic acid decreased the level of receptor mRNA on both plastic and collagen at each time but did not alter mRNA stability. For all treatments PTH/PTHrP receptor mRNA abundance, relative to glyceraldehyde-3-phosphate dehydrogenase, increased steadily over 24 h after subculture of cells. In contrast, PTHrP mRNA levels were reduced in cells on collagen, compared with plastic. PTH-stimulated cAMP levels of cells grown on collagen were increased compared with plastic at 24 h, but not earlier. Consistent with the mRNA data, retinoic acid decreased the amplitude of cAMP responses in cells on plastic and collagen. There was no evidence for changes in adenylate cyclase per se, since forskolin-induced cAMP levels did not change with either treatment. This study shows that known modulators of osteoblast maturation also affect signal transduction in these cells by regulating gene expression of the PTH/PTHrP receptor as well as the PTHrP ligand. Journal of Endocrinology (1996) 150, 299–308

scholarly journals The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts.

1994 ◽  
Vol 5 (4) ◽  
pp. 439-453 ◽  
Author(s):  
P Tremble ◽  
R Chiquet-Ehrismann ◽  
Z Werb
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Type I Collagen ◽  
Serum Proteins ◽  
Synovial Fibroblasts ◽  
Type I ◽  
Mixed Substrate ◽  
Amino Terminal ◽  
Collagenase Gene ◽  
In Cells

Tenascin (TN) is a large oligomeric glycoprotein that is present transiently in the extracellular matrix (ECM) of cells and is involved in morphogenetic movements, tissue patterning, and tissue repair. It has multiple domains, both adhesive and anti-adhesive, that interact with cells and with fibronectin (FN) and other ECM macromolecules. We have studied the consequences of the interaction of TN with a FN matrix on gene expression in rabbit synovial fibroblasts. Fibroblasts plated on a mixed substrate of FN and TN, but not on FN alone, upregulated synthesis of four genes: collagenase, stromelysin, the 92-kDa gelatinase, and c-fos. Although the fibroblasts spread well on both FN and FN/TN substrates, nuclear c-Fos increased within 1 h only in cells that were plated on FN/TN. TN did not induce the expression of collagenase in cells plated on substrates of type I collagen or vitronectin (VN). Moreover, soluble TN added to cells adhering to a FN substrate or to serum proteins had no effect, suggesting that TN has an effect only in the context of mixed substrates of FN and TN. Collagenase increased within 4 h of plating on a FN/TN substrate and exhibited kinetics similar to those for induction of collagenase gene expression by signaling through the integrin FN receptor. Arg-Gly-Asp peptide ligands that recognize either the FN receptor or the VN receptor and function-perturbing anti-integrin monoclonal antibodies diminished the interaction of fibroblasts with a mixed substrate of FN, TN, and VN, but had no effect on the adhesion of fibroblasts to a substrate of FN and VN, suggesting that both receptors recognize the complex. Anti-TN68, an antibody that recognizes an epitope in the carboxyl-terminal type III repeats involved in the interaction of TN with both FN and cells, blocked the inductive effect of the FN/TN substrate, whereas anti-TNM1, an antibody that recognizes an epitope in the amino-terminal anti-adhesive region of epidermal growth factor-like repeats, had no effect. These data suggest that transient alteration of the composition of ECM by addition of proteins like TN may regulate the expression of genes involved in cell migration, tissue remodeling, and tissue invasion, in regions of tissue undergoing phenotypic changes.

scholarly journals The Dynamic Interaction between Extracellular Matrix Remodeling and Breast Tumor Progression

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1046
Author(s):  
Jorge Martinez ◽  
Patricio C. Smith
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Cell Metabolism ◽  
Breast Tumors ◽  
Extracellular Matrix Remodeling ◽  
Type I ◽  
Matrix Remodeling ◽  
Desmoplastic Reaction ◽  
The Impact

Desmoplastic tumors correspond to a unique tissue structure characterized by the abnormal deposition of extracellular matrix. Breast tumors are a typical example of this type of lesion, a property that allows its palpation and early detection. Fibrillar type I collagen is a major component of tumor desmoplasia and its accumulation is causally linked to tumor cell survival and metastasis. For many years, the desmoplastic phenomenon was considered to be a reaction and response of the host tissue against tumor cells and, accordingly, designated as “desmoplastic reaction”. This notion has been challenged in the last decades when desmoplastic tissue was detected in breast tissue in the absence of tumor. This finding suggests that desmoplasia is a preexisting condition that stimulates the development of a malignant phenotype. With this perspective, in the present review, we analyze the role of extracellular matrix remodeling in the development of the desmoplastic response. Importantly, during the discussion, we also analyze the impact of obesity and cell metabolism as critical drivers of tissue remodeling during the development of desmoplasia. New knowledge derived from the dynamic remodeling of the extracellular matrix may lead to novel targets of interest for early diagnosis or therapy in the context of breast tumors.

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