scholarly journals Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells.

1994 ◽  
Vol 126 (1) ◽  
pp. 259-270 ◽  
Author(s):  
N Busso ◽  
S K Masur ◽  
D Lazega ◽  
S Waxman ◽  
L Ossowski
Keyword(s):  
Signal Transduction ◽  
Cell Line ◽  
Solid Phase ◽  
Shape Change ◽  
Epithelial Cell Line ◽  
Cell Fractionation ◽  
Western Blots ◽  
Glycosyl Phosphatidylinositol ◽  
Cell Lysates ◽  
Pkc Epsilon

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR-complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down-modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.

scholarly journals Bax gene expression alters Ca2+ signal transduction without affecting apoptosis in an epithelial cell line

Oncogene ◽  
2002 ◽  
Vol 21 (17) ◽  
pp. 2762-2767 ◽  
Author(s):  
Sonomi Aiba-Masago ◽  
Xiao-bing Liu ◽  
Rejei Masago ◽  
Norma Vela-Roch ◽  
Fabio Jimenez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Epithelial Cell ◽  
Cell Line ◽  
Epithelial Cell Line ◽  
Bax Gene

scholarly journals Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions

2015 ◽  
Vol 309 (12) ◽  
pp. C785-C798 ◽  
Author(s):  
Chin-Rang Yang ◽  
Pumipat Tongyoo ◽  
Milad Emamian ◽  
Pablo C. Sandoval ◽  
Viswanathan Raghuram ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Cell Line ◽  
Collecting Duct ◽  
Mass Spectrometry Data ◽  
Epithelial Cell Line ◽  
Proteomic Profiling ◽  
Western Blots ◽  
Sds Page ◽  
Public Data

The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out “deep” proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base ( https://helixweb.nih.gov/ESBL/Database/mpkFractions/ ). The mass spectrometry data were mapped back to their gel slices to generate “virtual Western blots” for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa.

Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line

1994 ◽  
Vol 107 (9) ◽  
pp. 2439-2448
Author(s):  
L. Rochwerger ◽  
S. Dho ◽  
L. Parker ◽  
J.K. Foskett ◽  
M. Buchwald
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Steroid Hormones ◽  
Primary Cultures ◽  
T Antigen ◽  
Epithelial Cell Line ◽  
Normal Female ◽  
Western Blots ◽  
Uterine Epithelial Cell

We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogen-coated filters was permeabilized with nystatin, a forskolin-stimulated Cl- permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogen-regulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.

Transmembrane Gla Protein 4 as a Novel Modulator of ERK2.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 544-544 ◽  
Author(s):  
Fayaz R. Khazi ◽  
Kirk C. Chu ◽  
Katherine A. High
Keyword(s):  
Signal Transduction ◽  
Cell Line ◽  
Cell Lines ◽  
Vitamin K ◽  
Cellular Localization ◽  
Mapk Signaling ◽  
Signaling Cascades ◽  
Type I ◽  
Cell Lysates ◽  
Signal Transduction Proteins

Abstract Transmembrane Gla protein 4 (TMG4) is a single pass, type I transmembrane protein with a vitamin-K dependent γ-carboxy-glutamic acid-rich domain (Kulman et al PNAS,2001). Bioinformatic analysis of the primary sequence indicates a very high degree of conservation (ca. 80%) among mammals and to-date no known functional roles have been assigned to this protein. In our efforts to elucidate molecular functions of TMG4, we first analyzed sub cellular localization of this protein. Microscopic analysis of HEK293T cells expressing a TMG4-GFP fusion construct showed juxtanuclear localization of the tagged protein at 24h post transfection. Staining with organelle specific fluorescent dyes revealed that the TMG4-GFP primarily localizes to the trans Golgi network in the cell. Recently, there have been increasing number of reports of golgi-localized proteins participating in compartmentalized signal transduction pathways and affecting cell differentiation and proliferation by activating major signaling cascades like the Ras-Raf/MAPK signaling cascades. Moreover, the transmembrane localization and the current lack of evidence for a direct role for TMG4 in the coagulation pathways encouraged us to hypothesize that TMG4 may play a role in signal transduction. To test this we generated a TMG4 over-expressing cell line (high-TMG4) by stably transfecting HEK293T cells with a CMV-promoter driven TMG4 plasmid , and a TMG4 knock-down cell line (low-TMG4) by stable transfections with a TMG4 short-hairpin RNA (shRNA) expression vector. The levels of TMG4 gene expression were confirmed in both the cell lines by RNA and protein expression analysis. The low-TMG cells showed >90% knockdown of TMG4 in comparison to untransfected control cells. The cells were grown under standard tissue culture conditions for 48h after which they were harvested. Total cell lysates from the cell lines were analyzed by western blotting using primary antibodies against a panel of signal transduction proteins representing major signaling pathways including threonine kinase, tyrosine kinase and ERK2. The results showed no significant changes in activation of signal transduction proteins tested except for extracellular-signal-regulated kinase (ERK) -2. The high-TMG cells showed a down modulation of phosphorylated ERK2 while the low-TMG4 cell lysates showed a 500-fold up regulation of phosphorylated ERK2 when compared to the levels in the low-TMG cells. Our observations were confirmed visually by assaying the cells for intracellular localization of ERK2. We used immuno-fluorescent staining to show that TMG4 levels regulate ERK2 activation based on TMG4 dependent translocation of ERK2 using anti phospho-ERK2 and anti-ERK2 antibodies. More than 85% of high-TMG4 cells showed extra nuclear localization of ERK2 while phosphorylated ERK2 was localized in the nucleus in the >80% of low-TMG4 cells. Thus there is an inverse correlation between TMG4 levels and ERK2 phosphorylation. We conclude that TMG4 is a molecular modulator of ERK2. Whether TMG4 has a direct interaction or affects ERK2 indirectly is under investigation. These studies suggest novel roles for vitamin-K dependent Gla-proteins in regulating thrombopoietin dependent megakaryocytic differentiation, platelet activation and myelopoiesis and myeloproliferation.

Ultrastructure of NPC/HK1 cells heterotransplanted in athymic nude mice

1982 ◽  
Vol 40 ◽  
pp. 236-237
Author(s):  
E.C. Chew ◽  
C.L. Li ◽  
D.P. Huang ◽  
H.C. Ho ◽  
L.S. Mak ◽  
...  
Keyword(s):  
Cell Line ◽  
Nude Mice ◽  
Biopsy Specimen ◽  
Epithelial Cell Line ◽  
Present Communication ◽  
Recurrent Tumour ◽  
Athymic Nude Mice ◽  
Cell Line Establishment ◽  
Fine Structures ◽  
Well Differentiated

An epithelial cell line, NPC/HK1, has recently been established from a biopsy specimen of a recurrent tumour of the nasopharynx which was histologically diagnosed as a moderately to well differentiated squamous cell carcinoma. A definite decrease in the amount of tonofilaments and desmosomes in the NPC/HK1 cells during the cell line establishment was observed. The present communication reports on the fine structures of the NPC/HK1 cells heterotraneplanted in athymic nude mice.

An Established Epithelial Cell Line (NPC/HK1) from a Nasopharyngeal Carcinoma - II. A Sem Study

1982 ◽  
Vol 40 ◽  
pp. 224-225
Author(s):  
Li C.L. ◽  
Chew E.C. ◽  
Huang D.P. ◽  
Ho H.C. ◽  
Mak L.S. ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Epithelial Cell ◽  
Surface Morphology ◽  
Cell Line ◽  
Epithelial Cell Line ◽  
Present Communication ◽  
Cells In Culture ◽  
Sem Study ◽  
Well Differentiated

An epithelial cell line, NPC/HK1, has recently been successfully established from a nasopharyngeal carcinoma of the moderately to well differentiated squamous type. The present communication reports on the surface morphology of the NPC/HK1 cells in culture.

A New Human Breast Tumor Cell Line

1975 ◽  
Vol 33 ◽  
pp. 388-389
Author(s):  
R.E. Nordquist ◽  
R.M. Wasik ◽  
P.J. Riggs ◽  
P.L. Munson ◽  
F.B. Schafer
Keyword(s):  
Electron Microscopy ◽  
Cell Line ◽  
Calf Serum ◽  
Tumor Cell Line ◽  
Electron Microscopic Examination ◽  
Epithelial Cell Line ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Excised Tissue ◽  
Caucasian Female

An infiltrating ductal cell carcinoma was removed from the breast of a postmenopausal Caucasian female. The excised tissue was divided into three parts; one part for electron microscopy, one part for tissue culture and the remainder frozen for immunological studies.The tissue for culture was minced finely with sterile razor blades and cultured in Falcon flasks containing Eagel's MEM supplemented with 10% heat denatured fetal calf serum. The tissue for electron microscopy was fixed in 6.25% glutaraldehyde in 0.1 M PO4 buffer plus 5% sucrose and postfixed in 1% OsO4 in the same buffer. The fixed tissue was dehydrated in graded ethanol and embedded in Spurr.The tissue which was cultured began to grow out after approximately six weeks and became a continuous epithelial cell line which was designated BOT-2 (Breast Original Tumor). Electron microscopic examination revealed that these cells had epithelial characteristics, i.e. the presence of tonofilaments and well formed desmosomes.

Sign in / Sign up

Export Citation Format

Share Document