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PubMed1993

Regulation of NCAM by growth factors in serum-free myotube cultures.

Regulation of the neural cell adhesion molecule (NCAM) was examined in primary cultures of chick skeletal muscle grown in serum-free defined medium. Relative levels of NCAM (per microgram protein) increased 20-30% in myotubes grown on Matrigel, a reconstituted basement membrane preparation, compared to those grown on collagen; total NCAM levels on Matrigel were increased 40-55% due to the additional increase in total protein. A dose dependent increase in relative NCAM levels in myotubes grown on Matrigel in defined medium was observed with the addition of adsorbed horse serum, while relative NCAM levels in myotubes grown on collagen were unaffected by altering the serum concentration. Thus, extracellular matrix molecules and soluble factors exert trophic effects on myotube NCAM expression. Similar developmental changes in the expression of the different molecular size forms of NCAM occurred in myotubes grown on collagen and Matrigel: levels of 150K and 135K Mr forms decreased during development, while 125K remained prominent in older myotubes. Relative NCAM levels were specifically enhanced 11-26% by several factors: nerve growth factor, thyroxine, insulin-like growth factor II, dibutyryl cyclic AMP, veratridine (a sodium ion channel agonist), and nisoldipine (a calcium ion channel agonist). Total protein and overall myotube development in serum-free cultures were enhanced by fetuin, insulin-like growth factor II, acidic fibroblast growth factor, calcitonin gene-related peptide, dibutyryl cyclic AMP, and veratridine. Thus, changes in extracellular matrix, intracellular calcium, and sodium ions, as well as extracellular trophic factors, such as nerve growth factor, thyroxine, and insulin-like growth factor II, may regulate muscle NCAM expression during embryonic development.

Acetylcholinesterase↗
PubMed1995

Isolation, culture, and characterization of equine oviduct epithelial cells in vitro.

Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17 beta or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers.

Animals↗
PubMed1994

Extracellular matrix components regulating glandular differentiation and the formation of basal lamina of a human pancreatic cancer cell line in vitro.

The interaction between the extracellular matrix and human tumor-cell clones S2-013 and S2-020, derived from a pancreatic cancer cell line (SUIT-2), was examined in vitro, using various cell differentiation-promoting matrices in two- and three-dimensional cultures. S2-013 cells (well-differentiated tubular adenocarcinoma in xenografts in nude mice) cultured in Matrigel formed glandular structures. Ultrastructural observation revealed a morphological polarity of cells and a distinct basal lamina. On the other hand, S2-020 cells (poorly differentiated tubular adenocarcinoma in xenografts) cultured in Matrigel formed neither glandular structures nor a basal lamina, but only cell aggregates. The morphology of these two sublines cultured in Matrigel expressed the histological degree of differentiation which they presented in nude mice. In contrast, in type I collagen gel, S2-013 cells formed glandular structures without a basal lamina, and in soft agar, they were able to form neither glandular structures nor a basal lamina. S2-020 cells cultured in type I collagen gel or soft agar formed the same simple cell aggregates as in Matrigel. Matrices used in a three-dimensional culture influenced the degree of differentiation in S2-013 cells but had no effect on the morphological differentiation in S2-020 cells. To detect the factors which induce basal lamina formation, S2-013 cells were cultured on a microporous membrane coated with extra-cellular matrix components such as laminin, type IV collagen, and fibronectin. S2-013 cells formed a basal lamina only on the laminin. These cell lines may be useful in investigating the mechanisms regulating the formation of glandular structures and basal lamina.

Adenocarcinoma↗
PubMed1995

Substrate modulation of morphology, growth, and tear protein production by cultured human lacrimal gland epithelial cells.

Human lacrimal gland biopsies were serially incubated in chelating and enzymatic solutions. Viable single cells were evaluated for outgrowth in growth factor enriched medium using the following culture substrates: Matrigel, Type I collagen gel with or without fibroblasts, and plastic. Each epithelial outgrowth was characterized morphologically and immunohistochemically, and their growth and viability were examined by BrdU labeling and a quantitative cell viability assay. Synthesized proteins were evaluated by ELISA, SDS-PAGE, and 14C-labeled amino acid incorporation. Lacrimal gland epithelial cells plated on Matrigel formed clusters with central cavities that contained lactoferrin, mimicking acinar complexes in vivo. Cells plated on collagen gel or collagen gel containing fibroblasts formed islands or a monolayer, and lactoferrin was detected in incomplete cavities of epithelia on the latter substrate. Epithelial cells plated on plastic formed a monolayer and cellular expression of lactoferrin was weak and sporadic. Cellular release of the lactoferrin measured by ELISA supported the results of immunohistochemistry. Cells grown on plastic had the highest proliferative rate, whereas those grown on Matrigel showed the lowest proliferative rate. These results indicate that different substrates modulate lacrimal gland epithelial cell morphology, proliferative rate, and production of the tear protein lactoferrin. Matrigel promotes acinar differentiation to a greater extent than collagen gel and plastic. Incorporation of fibroblasts in collagen gel substrate promotes significant effects on growth and differentiation.

Adult↗
PubMed1996

Inhibition of endothelial cell differentiation on a glycosylated reconstituted basement membrane complex.

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.

Albumins↗
PubMed1996

Extracellular matrix penetration by epithelial cells is influenced by quantitative changes in basement membrane components and growth factors.

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substrate in vitro to form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin-nidogen complex to the substrate or by addition of TGFbeta2 to the culture medium, but not TGFbeta1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.

Animals↗
PubMed1999

An in vitro tubule assay identifies HGF as a morphogen for the formation of seminiferous tubules in the postnatal mouse testis.

We have been working with a recently immortalized Sertoli cell line, SF7, that appears to produce sleeves, or hollow tubules, when cultivated on a layer of growth factor-reduced Matrigel (GFR-Matrigel) in medium supplemented with serum. We tried to determine which components of GFR-Matrigel and serum provide the environment needed for tubule formation. While laminin and collagen IV were essential for the formation of flat cords, none of the basement membrane constituents, when taken alone or in combination, would support the formation of tubules in minimal culture medium. Moreover, none of the growth factors present in GFR-Matrigel could induce tubulogenesis. Recently, much attention has been focused upon the role of hepatocyte growth factor (HGF) and its receptor c-met in the induction of tubulogenesis by epithelial cells. Therefore, we investigated the expression of HGF/c-met in the mouse testis at different postnatal stages and in the adult and evaluated the contribution of HGF/c-met in the production of Sertoli cell tubules by SF7 and primary Sertoli cells in vitro. Our results confirm that laminin and collagen IV are essential for the formation of testicular cords and reveal that HGF/c-met are necessary for the further remodeling of these cords into tubules.

Animals↗
PubMed2000

Nitric oxide induces angiogenesis and upregulates alpha(v)beta(3) integrin expression on endothelial cells.

Nitric oxide (NO) has been implicated as a mediator of angiogenesis. However, its precise role in angiogenesis and its mechanism of action have not been established. We performed in vivo and in vitro angiogenesis assays using NO donor S-nitroso-N-acetylpenicillamine (SNAP) and NO synthase inhibitor N-iminoethyl-l-ornithine (L-NIO). SNAP significantly increased and L-NIO significantly suppressed capillary ingrowth into subcutaneously implanted Matrigel plugs in mice. For the in vitro angiogenesis assay, human umbilical vein endothelial cells (HUVECs) (4 x 10(4) cells/well) were treated with placebo, SNAP (100 microM), or L-NIO (100 microM) and cultured on Matrigel for 18 h. The typical capillary networks formed on Matrigel by HUVECs as a result of cell migration and differentiation were quantified by computer-assisted image analysis as a measure of angiogenesis. Treatment of HUVECs with SNAP significantly increased the capillary network area compared with control, 8701 +/- 693 vs 6258 +/- 622 area units (P < 0.05), whereas L-NIO significantly decreased the capillary area (4540 +/- 342, P < 0.05). Furthermore, we have shown with a blocking monoclonal antibody that formation of capillary networks on Matrigel is mediated by the functional expression of the alpha(v)beta(3) integrin, which plays a role in facilitating endothelial cell adhesion to basement membrane matrix and endothelial cell migration. After an 18-h culture, flow cytometry revealed that SNAP significantly upregulated and L-NIO significantly downregulated in a concentration-dependent manner alpha(v)beta(3) integrin expression on endothelial cells. In conclusion, NO induces angiogenesis in vivo and in vitro by promoting endothelial cell migration and differentiation into capillaries. One possible mechanism might involve the upregulation of alpha(v)beta(3) integrin on endothelial cells, a critical mediator of cell-matrix adhesion and migration.

Animals↗
PubMed1991

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix.

The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells.

Animals↗
PubMed1991

Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro.

The extracellular matrix, particularly basement membranes, plays an important role in angiogenesis (blood vessel formation). Previous work has demonstrated that a basement membranelike substrate (Matrigel) induces human umbilical vein endothelial cells to rapidly form vessel-like tubes (Kubota, et al., 1988; Grant et al., 1989b); however, the precise mechanism of tube formation is unclear. Using this in vitro model, we have investigated morphologic changes occurring during tube formation and the cytoskeletal and protein synthesis requirements of this process. Electron microscopy showed that endothelial cells attach to the Matrigel surface, align, and form cylindrical structures that contain a lumen and polarized cytoplasmic organelles. The cytoskeleton is reorganized into bundles of actin filaments oriented along the axis of the tubes and is located at the periphery of the cells. The addition of colchicine or cytochalasin D blocked tube formation, indicating that both microfilaments and microtubules are involved in this process. Cycloheximide blocked tube formation by 100%, indicating that the process also required protein synthesis. In particular, collagen synthesis seems to be required for tube formation because cis-hydroxyproline inhibited tube formation, whereas either the presence of ascorbic acid or the addition of exogenous collagen IV to the Matrigel increased tube formation. Our results indicate that endothelial cell attachment to Matrigel induces the reorganization of the cytoskeleton and elicits the synthesis of specific proteins required for the differentiated phenotype of the cells.

Ascorbic Acid↗
PubMed1991

Proliferation and differentiation of mouse uterine epithelial cells in primary serum-free culture: estradiol-17 beta suppresses uterine epithelial proliferation cultured on a basement membrane-like substratum.

The effects of different substrata and estradiol-17 beta (E2) on proliferation and differentiation of mouse uterine epithelial cells was examined in a serum-free primary culture system. When cultured on rat-tail collagen gels, the epithelial cells rapidly increased in number to form a simple squamous cell layer that exhibited a relatively undifferentiated state (a few short microvilli, no secretory granules, and poorly developed endoplasmic reticulum). Addition of E2 into the culture medium did not affect the proliferation of epithelial cells on collagen gel. Uterine epithelial cells grown on a reconstituted basement membrane-like substratum (Matrigel) formed a simple columnar/cuboidal cell layer exhibiting fully developed characteristics (many long microvilli, many secretory granules, and fully developed endoplasmic reticulum). Examination of epithelial proliferation by counting substratum-attached cell number revealed only a slow increase in cell growth on Matrigel, and E2 did not significantly affect it. However, measurement of proliferating cells by labeling cells with 5-bromo-2'-deoxyuridine revealed that cells on Matrigel were replicating and that E2 (10(-7) to 10(-11) M) actually significantly suppressed epithelial proliferation. However, there was not an effect of E2 on total cell number, indicating that the cells in control medium replicate faster and detach more readily from the substratum than those in E2-supplemented medium on Matrigel. Thus, it is probable that E2 significantly reduces the rate of cell detachment from the substratum, which may mimic the in vivo condition where significant decrease in apoptosis or cell death is induced by E2.

Animals↗
PubMed1993

Rat Sertoli cell aromatase cytochrome P450: regulation by cell culture conditions and relationship to the state of cell differentiation.

Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Effect of spatial arrangement of the basement membrane on cultured pleomorphic adenoma cells. Study by immunocytochemistry and electron and confocal microscopy.

In a cell line from human pleomorphic adenoma (AP2 cells) we studied the response of these cells to basement membrane proteins. The culture was characterized as myoepithelial-like by transmission electron microscopy and immunocytochemistry. AP2 cells were grown in contact with a reconstituted basement membrane (Matrigel). Cells grown on Matrigel showed conspicuous phenotypic alterations, depending on how the substrate was applied. Cells grown on the top of Matrigel developed a dendritic phenotype, exhibiting thin, long and intercommunicating cytoplasmic extensions resembling normal myoepithelial cells. Cells grown inside Matrigel formed multi-layered clusters. Light, confocal and transmission electron microscopy showed that these clusters were formed by double-layered epithelioid cells delimiting luminal spaces. The cells facing the lumen were cuboidal, showing microvilli at the apical plasmalemmal and junctional complexes. The spatial arrangement of basement membrane is a key modulator of morphogenetic changes and cytodifferentiation of tumour myoepithelial cell lineage in culture.

Adenoma, Pleomorphic↗
PubMed1998

Effects of tri-iodothyronine (T3), insulin, insulin-like growth factor I (IGF-I) and transforming growth factor beta1 (TGFbeta1) on the proportion of insulin cells in cultured embryonic chick pancreas.

With a view to ultimately identifying factors involved in the development of pancreatic insulin cells, we have cultured dorsal pancreatic buds from 5-day chick embryos on a basement membrane matrix (Matrigel) in a serum-free medium supplemented with selected factors. The endodermal components of the buds were freed of almost all the mesenchyme so as to eradicate as much as possible of this source of some such factors. In 7-day cultures, insulin and glucagon cells were demonstrated immunocytochemically; numbers of insulin cells were expressed as a percentage of insulin plus glucagon cell counts. Our standard medium contained insulin. Addition of tri-iodothyronine to this medium did not increase the proportion of insulin cells, but in combination with raised concentrations of glucose and essential amino acids it improved somewhat the marked increase previously recorded for these nutrient conditions. Omission of insulin from the standard medium greatly reduced the proportion of these cells; substitution of insulin by insulin-like growth factor I increased the proportion considerably more than did insulin. To test for an overall effect of growth factors, explants were cultured in standard medium on Matrigel containing reduced amounts of growth factors: the proportion of insulin cells proved to be increased over that reached on normal Matrigel. The suspicion that transforming growth factor beta1, a component of Matrigel, might act to reduce the proportion of insulin cells was tested and found to be correct. It is suggested that the different factors studied here may affect either or both of proliferation and determination in the differentiation pathway of insulin vis-à-vis glucagon cells.

Animals↗
PubMed2007

Evidence for the involvement of p38 MAP kinase in the action of the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

AIMS: DMXAA (AS1404), a small-molecule vascular disrupting agent that has now completed Phase II clinical trial, induces endothelial cell apoptosis, increased vascular permeability and decreased tumour blood flow in vivo. Its action is incompletely understood and we wished to develop an in vitro system to study its effects. METHODS: Human tumour cell lines developed from aggressive tumours were grown on Matrigel to simulate a tumour microenvironment. Cells were analysed by light microscopy and by gene expression profiling. RESULTS: Several cell lines formed networks when grown on Matrigel and the NZM7 melanoma cell line was chosen for further study. Addition of DMXAA at a clinically achievable concentration (30 microg/mL) prevented network formation, but co-addition of SB203580 (10 microM), a selective inhibitor of p38 MAP kinase, reversed the effect of DMXAA and restored network formation. Analysis of expression genes for endothelial and related functions showed that cells growing on Matrigel expressed a pattern similar to that of NZM7 cells growing as xenografts in vivo but different from that of cells grown on standard tissue culture plates. Addition of DMXAA resulted in the inhibition of expression of several genes including the transcriptional activator Ets1 and matrix metalloproteinase-2 (MMP2), but co-addition of SB203580 did not reverse these effects of DMXAA on gene expression. CONCLUSION: The results suggest that p38 MAP kinase plays an important role in the action of DMXAA and that growth of tumour cells on Matrigel provides a promising model for further studies on the action of this drug.

Angiogenesis Inhibitors↗
PubMedDate not supplied

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture.

The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 microM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 microM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.

Animals↗
PubMed1992

Glandular-like morphogenesis of the human submandibular tumor cell line A253 on basement membrane components.

We have studied the interaction of a human tumor cell line, A253, derived from a submandibular gland carcinoma with a differentiation promoting reconstituted basement membrane extract, Matrigel. When cultured on plastic, these cells maintain a flat, cobblestone, epithelial morphology. On Matrigel, A253 cells initially form a honeycomb network of cords of cells which subsequently thickens. With time, these cords of cells become discontinuous and blunted, whereupon multilobular clusters of cells develop. These clusters possess a lumen with polarized, PAS(+) cells containing numerous desmosomes and an abundance of glycogen. Culture of the cells on laminin, the most abundant protein found in Matrigel, also induces this morphologic differentiation. Using synthetic laminin-derived peptides, the biologically active IKVAV-containing site of laminin was most active in attachment assays, as well as in inhibiting glandular-like morphogenesis when added to the media of cells cultured on Matrigel. Antibodies to the cell surface 67- and 32-kDa laminin binding proteins partially inhibited the glandular-like morphogenesis, suggesting that multiple interactions with laminin are likely required for the differentiation process. Our data demonstrate that A253 cells can undergo glandular-like morphogenesis on basement membrane and that laminin appears to be the major initiating factor.

Blotting, Western↗
PubMed1993

Extracellular matrix composition influences insulinlike growth factor I receptor expression in rat IEC-18 cells.

BACKGROUND: The composition of the extracellular matrix (ECM) as well as insulinlike growth factor I (IGF-I) receptor density vary along the crypt-villus axis. We determined whether components of the ECM influence IGF-I receptor expression in IEC-18 rat small intestine crypt cells. METHODS: IEC-18 cells were cultured on plastic, collagen type IV, Matrigel, and laminin at the plateau and proliferative growth phases. Receptor affinity (Kd) and number (Bmax) were determined by competitive binding of 125I-IGF-I in the presence of increasing concentrations of unlabeled IGF-I. Receptor isolation was performed by affinity cross linking. Messenger RNA (mRNA) for IGF-I receptor was quantified by Northern analysis. RESULTS: Specific binding of IGF-I > IGF-II > insulin was observed. A 130,000-molecular weight protein was identified by cross-linking, consistent with the alpha subunit of the IGF-I receptor. Scatchard analysis revealed no effect of ECM on IGF-I binding affinity. In contrast, the Bmax was 18% lower for plateau-phase cells cultured on Matrigel vs. plastic and was 42% lower for cells cultured on laminin vs. collagen type IV. The Bmax for proliferative growth phase cells was decreased when cultured on Matrigel vs. plastic and was 10-fold less than for cells cultured at the plateau growth phase. Northern analysis revealed that IEC-18 cells cultured on Matrigel had less mRNA for IGF-I receptor than cells cultured on plastic. CONCLUSIONS: The rate of cell proliferation and the composition of the ECM influence IGF-I receptor expression in IEC-18 cells.

Animals↗