scholarly journals Cell Elongation Induces Laminin α2 Chain Expression in Mouse Embryonic Mesenchymal Cells

1999 ◽  
Vol 147 (6) ◽  
pp. 1341-1350 ◽  
Author(s):  
Nand K. Relan ◽  
Yan Yang ◽  
Safedin Beqaj ◽  
Jeffrey H. Miner ◽  
Lucia Schuger
Keyword(s):  
Cell Shape ◽  
Contractile Activity ◽  
Congenital Muscular Dystrophy ◽  
Mesenchymal Cell ◽  
Specific Protein ◽  
Mesenchymal Cells ◽  
Bronchial Smooth Muscle ◽  
Functional Studies ◽  
Low Levels ◽  
Dy Mouse

Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) α2 chain not present in round mesenchymal cells. LM α2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM β1 and γ1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM α2. Dy/dy mice express very low levels of LM α2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM α-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM α2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.

scholarly journals High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis

2002 ◽  
Vol 156 (5) ◽  
pp. 893-903 ◽  
Author(s):  
Safedin Beqaj ◽  
Sandhya Jakkaraju ◽  
Raymond R. Mattingly ◽  
Desi Pan ◽  
Lucia Schuger
Keyword(s):  
Smooth Muscle ◽  
Serum Response Factor ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
Small Gtpase ◽  
Cell Phenotype ◽  
Rhoa Activity ◽  
Functional Studies ◽  
Undifferentiated Mesenchymal Cell ◽  
Main Components

Round embryonic mesenchymal cells have the potential to differentiate into smooth muscle (SM) cells upon spreading/elongation (Yang, Y., K.C. Palmer, N. Relan, C. Diglio, and L. Schuger. 1998. Development. 125:2621–2629; Yang, Y., N.K. Relan, D.A. Przywara, and L. Schuger. 1999. Development. 126:3027–3033; Yang, Y., S. Beqaj, P. Kemp, I. Ariel, and L. Schuger. 2000. J. Clin. Invest. 106:1321–1330). In the developing lung, this process is stimulated by peribronchial accumulation of laminin (LN)-2 (Relan, N.K., Y. Yang, S. Beqaj, J.H. Miner, and L. Schuger. 1999. J. Cell Biol. 147:1341–1350). Here we show that LN-2 stimulates bronchial myogenesis by down-regulating RhoA activity. Immunohistochemistry, immunoblotting, and reverse transcriptase–PCR indicated that RhoA, a small GTPase signaling protein, is abundant in undifferentiated embryonic mesenchymal cells and that its levels decrease along with SM myogenesis. Functional studies using agonists and antagonists of RhoA activation and dominant positive and negative plasmid constructs demonstrated that high RhoA activity was required to maintain the round undifferentiated mesenchymal cell phenotype. This was in part achieved by restricting the localization of the myogenic transcription factor serum response factor (SRF) mostly to the mesenchymal cell cytoplasm. Upon spreading on LN-2 but not on other main components of the extracellular matrix, the activity and level of RhoA decreased rapidly, resulting in translocation of SRF to the nucleus. Both cell elongation and SRF translocation were prevented by overexpression of dominant positive RhoA. Once the cells underwent SM differentiation, up-regulation of RhoA activity induced rather than inhibited SM gene expression. Therefore, our studies suggest a novel mechanism whereby LN-2 and RhoA modulate SM myogenesis.

scholarly journals EpCAM promotes endosomal modulation of the cortical RhoA zone for epithelial organization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cécile Gaston ◽  
Simon De Beco ◽  
Bryant Doss ◽  
Meng Pan ◽  
Estelle Gauquelin ◽  
...  
Keyword(s):  
Cell Shape ◽  
Specific Protein ◽  
Cell Polarization ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Endosomal Trafficking ◽  
Transient Zone ◽  
And Behavior ◽  
Fine Tune

AbstractAt the basis of cell shape and behavior, the organization of actomyosin and its ability to generate forces are widely studied. However, the precise regulation of this contractile network in space and time is unclear. Here, we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility. We show that EpCAM is required for stress fiber generation and front-rear polarity acquisition at the single cell level. In fact, EpCAM participates in the remodeling of a transient zone of active RhoA at the cortex of spreading epithelial cells. EpCAM and RhoA route together through the Rab35/EHD1 fast recycling pathway. This endosomal pathway spatially organizes GTP-RhoA to fine tune the activity of actomyosin resulting in polarized cell shape and development of intracellular stiffness and traction forces. Impairment of GTP-RhoA endosomal trafficking either by silencing EpCAM or by expressing Rab35/EHD1 mutants prevents proper myosin-II activity, stress fiber formation and ultimately cell polarization. Collectively, this work shows that the coupling between co-trafficking of EpCAM and RhoA, and actomyosin rearrangement is pivotal for cell spreading, and advances our understanding of how biochemical and mechanical properties promote cell plasticity.

scholarly journals Characterization of adenylyl cyclase isoforms in rat peripheral pulmonary arteries

2001 ◽  
Vol 280 (6) ◽  
pp. L1359-L1369 ◽  
Author(s):  
Karen B. Jourdan ◽  
Nicola A. Mason ◽  
Lu Long ◽  
Peter G. Philips ◽  
Martin R. Wilkins ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
Pulmonary Arteries ◽  
Bronchial Epithelium ◽  
Rt Pcr ◽  
Bronchial Smooth Muscle ◽  
Functional Studies ◽  
Kinase C ◽  
Rat Lungs

Activation of adenylyl cyclase (AC), of which there are 10 diversely regulated isoforms, is important in regulating pulmonary vascular tone and remodeling. Immunohistochemistry in rat lungs demonstrated that AC2, AC3, and AC5/6 predominated in vascular and bronchial smooth muscle. Isoforms 1, 4, 7, and 8 localized to the bronchial epithelium. Exposure of animals to hypoxia did not change the pattern of isoform expression. RT-PCR confirmed mRNA expression of AC2, AC3, AC5, and AC6 and demonstrated AC7 and AC8 transcripts in smooth muscle. Western blotting confirmed the presence of AC2, AC3, and AC5/6 proteins. Functional studies provided evidence of cAMP regulation by Ca2+ and protein kinase C-activated but not Gi-inhibited pathways, supporting a role for AC2 and a Ca2+-stimulated isoform, AC8. However, NKH-477, an AC5-selective activator, was more potent than forskolin in elevating cAMP and inhibiting serum-stimulated [3H]thymidine incorporation, supporting the presence of AC5. These studies demonstrate differential expression of AC isoforms in rat lungs and provide evidence that AC2, AC5, and AC8 are functionally important in cAMP regulation and growth pathways in pulmonary artery myocytes.

Acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) activity distribution pattern in early developing chick limbs

Development ◽  
1985 ◽  
Vol 86 (1) ◽  
pp. 89-108
Author(s):  
Carla Falugi ◽  
Margherita Raineri
Keyword(s):  
Plasma Membrane ◽  
Basal Lamina ◽  
Ache Activity ◽  
Quantitative Methods ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
Regional Localization ◽  
Stage Of Development ◽  
Limb Morphogenesis ◽  
Cell Processes

The distribution of acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) activities was studied by histochemical, quantitative and electrophoretical methods during the early development of chick limbs, from stage 16 to stage 32 H.H. (Hamburger & Hamilton, 1951). By quantitative methods, true AChE activity was found, and increased about threefold during the developmental period, together with a smaller amount of BuChE which increased more rapidly in comparison with the AChE activity from stage 25 to 32 H.H. Cholinesterase activity was histochemically localized mainly in interacting tissues, such as the ectoderm (including the apical ectodermal ridge) and the underlying mesenchyme. True AChE was histochemically localized around the nuclei and on the plasma membrane of ectodermal (including AER) and mesenchymal cells, and at the plasma membrane of mesenchymal cell processes reaching the basal lamina between the ectoderm and the mesenchyme. AChE together with BuChE activity was found in the basal lamina between the ectoderm and the mesenchyme, in underlying mesenchymal cells and in deeper mesenchymal cells, especially during their transformation into unexpressed chondrocytes. During limb morphogenesis, the cellular and regional localization of the enzyme activities showed variations depending on the stage of development and on the occurrence of interactions. The possibility of morphogenetic functions of the enzyme is discussed.

Fibrogenesis IV. Fibrosis and inflammatory bowel disease: cellular mediators and animal models

2000 ◽  
Vol 279 (4) ◽  
pp. G653-G659 ◽  
Author(s):  
Jolanta B. Pucilowska ◽  
Kristen L. Williams ◽  
P. Kay Lund
Keyword(s):  
Sulfonic Acid ◽  
Genetic Manipulation ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
Acute Injury ◽  
Ethanol Administration ◽  
Cell Phenotype ◽  
Trinitrobenzene Sulfonic Acid ◽  
Intestinal Fibrosis ◽  
Cellular Mediators

The cellular mediators of intestinal fibrosis and the relationship between fibrosis and normal repair are not understood. Identification of the types of intestinal mesenchymal cells that produce collagen during normal healing and fibrosis is vital for elucidating the answers to these questions. Acute injury may cause normal mesenchymal cells to convert to a fibrogenic phenotype that is not maintained during normal healing but may lead to fibrosis when inappropriately sustained. Proliferation of normal or fibrogenic mesenchymal cells may lead to muscularis overgrowth associated with fibrosis. The presence of increased numbers of vimentin-positive cells within fibrotic, hypertrophied muscularis in Crohn's disease suggests that changes in mesenchymal cell phenotype and number may indeed be associated with fibrosis. Fibrosis is induced in rats by peptidoglycan polysaccharides or trinitrobenzene sulfonic acid-ethanol administration, but inducing fibrosis in mice has been technically challenging. The development of current mouse models of colitis, such as dextran sodium sulfate or trinitrobenzene sulfonic acid-ethanol administration, into models of fibrosis will allow us to use genetic manipulation to study molecular mediators of fibrosis.

Functional studies of PLIM-1, a pollen-specific protein

1999 ◽  
pp. 139-148
Author(s):  
Å Eliasson ◽  
N. Gass ◽  
A. Steinmetz
Keyword(s):  
Specific Protein ◽  
Functional Studies

Skeletal muscle phenotype signaling with ex vivo endurance-type dynamic contractions in rat muscle

2021 ◽  
Author(s):  
Jesper Emil Jakobsgaard ◽  
Jacob Andresen ◽  
Frank V. de Paoli ◽  
Kristian Vissing
Keyword(s):  
Skeletal Muscle ◽  
Translation Initiation ◽  
Ex Vivo ◽  
Contractile Activity ◽  
Specific Protein ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Metabolic Signaling ◽  
Dynamic Endurance

Skeletal muscle phenotype may influence the response sensitivity of myocellular regulatory mechanisms to contractile activity. To examine this, we employed an ex vivo endurance-type dynamic contraction model to evaluate skeletal muscle phenotype-specific protein signaling responses in rat skeletal muscle. Preparations of slow-twitch soleus and fast-twitch extensor digitorum longus skeletal muscle from 4-wk old female Wistar rats were exposed to an identical ex vivo dynamic endurance-type contraction paradigm consisting of 40 minutes of stretch-shortening contractions under simultaneous low-frequency electrostimulation delivered in an intermittent pattern. Phosphorylation of proteins involved in metabolic signaling and signaling for translation initiation was evaluated at 0, 1, and 4 hours after stimulation by immunoblotting. For both muscle phenotypes, signaling related to metabolic events was upregulated immediately after stimulation, with concomitant absence of signaling for translation-initiation. Signaling for translation-initiation was then activated in both muscle phenotypes at 1-4 hours after stimulation, coinciding with attenuated metabolic signaling. The recognizable pattern of signaling responses support how our ex vivo dynamic muscle contraction model can be utilized to infer a stretch-shortening contraction pattern resembling stretch-shortening contraction of in vivo endurance exercise. Moreover, using this model, we observed that some specific signaling proteins adhering to metabolic events or to translation initation exhibited phosphorylation changes in a phenotype-dependent manner, whereas other signaling proteins exhibited phenotype-independent changes. These findings may aid the interpretation of myocellular signaling outcomes adhering to mixed muscle samples collected during human experimental trials.

Lymphocyte-Specific Protein 1 Expression in Eukaryotic Cells Reproduces the Morphologic and Motile Abnormality of NAD 47/89 Neutrophils

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4786-4795 ◽  
Author(s):  
Thomas H. Howard ◽  
John Hartwig ◽  
Casey Cunningham
Keyword(s):  
Actin Polymerization ◽  
Cell Function ◽  
Specific Protein ◽  
Polymorphonuclear Neutrophils ◽  
Actin Binding ◽  
Actin Network ◽  
Cell Surfaces ◽  
Actin Bundles ◽  
Mixed Polarity ◽  
Low Levels

Abstract Despite its name, the actin-binding protein lymphocyte-specific protein1 (LSP1) is found in all hematopoetic cells, and yet its role in cell function remains unclear. Recently, LSP1 was identified as the 47-kD protein overexpressed in the polymorphonuclear neutrophils of patients with a rare neutrophil disorder, neutrophil actin dysfunction with abnormalities of 47-kD and 89-kD proteins (NAD 47/89). These neutrophils are immotile, defective in actin polymerization in response to agonists, and display distinctive, fine, “hairlike” F-actin-rich projections on their cell surfaces. We now show that overexpression of LSP1 produces F-actin bundles that are likely responsible for the morphologic and motile abnormalities characteristic of the NAD 47/89 phenotype. Coincident with LSP1 overexpression, cells from each of several different eukaryotic lines, including a highly motile human melanoma line, develop hairlike surface projections that branch distinctively and contain F-actin and LSP1. The hairlike projections are supported at their core by thick actin bundles, composed of actin filaments of mixed polarity, which periodically anastomose to generate a branching structure. The motility of the melanoma cells is inhibited even at low levels of LSP1 expression. Therefore, these studies show that overexpression of LSP1 alone can recreate the morphologic and motile defects seen in NAD 47/89 and suggest that LSP1 is distinct from other known actin binding proteins in its effect on F-actin network structure.

Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA

2000 ◽  
Vol 279 (5) ◽  
pp. F866-F873 ◽  
Author(s):  
Omar F. Laterza ◽  
Lynn Taylor ◽  
Shashikala Unnithan ◽  
Ly Nguyen ◽  
Norman P. Curthoys
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Luciferase Activity ◽  
Specific Protein ◽  
Luciferase Reporter ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Functional Studies ◽  
Renal Gluconeogenesis ◽  
Shift Analysis ◽  
Nontranslated Region

Phosph oenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3′-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3′-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3′-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.

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