Substrate adhesion of rat hepatocytes: a comparison of laminin and fibronectin as attachment proteins.

Johansson, Staffan; Kjellén, Lena; Höök, Magnus; Timpl, Rupert

doi:10.1083/jcb.90.1.260

Cited by 155 publications

(52 citation statements)

References 21 publications

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“…In accordance with previous results (26), rat hepatocytes were found to attach to culture dishes coated with 20~g of fibronectin or laminin when incubated at 37°C (Fig. 1 A).…”

Section: Attachment Of Rat Hepatocytes To Fibronectin and Laminin At supporting

confidence: 77%

“…The inhibitory effect was specific for soluble fibronectin ; other proteins, i.e., laminin (26), collagen (Table I) Inhibition of cell attachment by soluble fibronectin . Cells were preincubated with soluble fibronectin as described in Materials and Methods and the time course of attachment to fibronectin substrates in the presence of 0.1 mg fibronectin/ml (") or 0 .1 mg heat denatured fibronectin/ml (~) at 37°C (A) and 4°C (8), were followed .…”

Section: Inhibition Of Cell Attachment By Fibronectin In Solutionmentioning

confidence: 99%

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Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin.

Johansson¹,

Höök

1984

The Journal of Cell Biology

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134

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We examined the mechanisms of cell attachment to fibronectin-coated substrates. Inhibition of cell attachment was obtained by species-specific antifibronectin antibodies, which presumably recognize a distinct antigenic structure in the protein located at, or in the immediate vicinity of, the cell-binding site. The inhibiting antibodies could be adsorbed on a column of Sepharose substituted with plasma fibronectin. The initial phase of cell attachment was also inhibited by addition of soluble fibronectin to the incubation medium in a reaction that exhibited specificity and concentration dependence. These data suggest that cell-binding sites are available in an active form on the surface of soluble fibronectin. However, the inhibitory effect of fibronectin was greatly enhanced by adding the protein together with heparin, heparan sulfate, collagen, or a fibronectin-binding collagen peptide (CB-7), which is consistent with an "activation" of fibronectin on binding to these matrix components. A similar activation of fibronectin was obtained by cleaving the protein with trypsin. We discuss these findings in relation to conformational rearrangements in the fibronectin molecule. Data is presented supporting a mechanism of cell attachment to fibronectin involving multiple weak interactions between cellular receptors and substrate molecules, although some steps in the attachment process appear to disobey the requirements for such a mechanism.

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“…In accordance with previous results (26), rat hepatocytes were found to attach to culture dishes coated with 20~g of fibronectin or laminin when incubated at 37°C (Fig. 1 A).…”

Section: Attachment Of Rat Hepatocytes To Fibronectin and Laminin At supporting

confidence: 77%

Section: Inhibition Of Cell Attachment By Fibronectin In Solutionmentioning

confidence: 99%

Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin.

Johansson¹,

Höök

1984

The Journal of Cell Biology

Self Cite

134

View full text Add to dashboard Cite

show abstract

“…dicated in the text. 3 ml of the cell suspensions (2-3 X 10' cells) were seeded on 60-mm plastic dishes that had been precoated with fibronectin (16,18,24,25). (Dishes were prepared by incubating them with 25 usg fibronectin in 2.5 ml DME for at least 1 h at 370C).…”

Section: Methodsmentioning

confidence: 99%

Role of insulin in lipoprotein secretion by cultured rat hepatocytes.

Patsch¹,

Franz²,

Schonfeld³

1983

J. Clin. Invest.

173

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A B S T R A C T To study the effect of insulin on lipoprotein synthesis and secretion by the liver, apoprotein and lipid levels were measured in primary rat liver cell cultures grown on fibronectin-coated dishes. Triglycerides, phospholipids, apoprotein (apo) B, apo-E, and apo-C-III3 all accumulated in culture media linearly for periods up to 20 h. During incubations, cellular triglyceride contents increased slightly, while cellular apoprotein and phospholipid contents remained constant. In the absence of insulin, rates of accumulation in media of triglycerides, apo-B, apo-C-III3, and apo-E were 2.5±0.3 Ag/mg and 33±5, 24±3, and 162±32 ng/mg cell protein per h, respectively. On gel permeation chromatography and density gradient ultracentrifugation, the majority of apoproteins in media were found to be associated with very low density lipoproteins (VLDL) and very little eluted or sedimented with albumin. Incubations in the presence of 50-800 MU/ml of insulin resulted in dose-dependent decreases of triglyceride, phospholipid, apo-B, and apo-E accumulation in the media, paralleled by increases in the cellular contents of these lipoprotein components. The inhibitory effects of insulin on secretion were reversible. Levels of apo-C-III3 and albumin were not affected by insulin. In addition to decreasing secretory rates, the proportion of apo-B, apo-E, and apo-C-III3 associated with VLDL also decreased after the addition of insulin. Concomitantly, the proportion of apo-B eluting with LDL and apo-C-1113, and apo-E eluting near albumin increased. Control experiments, in which exogenous 125I-VLDL or endogenously labeled [14C]VLDL were added to cultures, revealed that the insulin-induced differences in VLDL accumulation and the lipid association of media apoproteins were not due to differences in the processing of VLDL by cells cultured in the presence or absence of insulin. Therefore, it appears that insulin

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“…For transcriptional activity and mRNA levels, expression and, more importantly, that induction is initiated nt producing the most intense signal on resulting at the transcriptional level by an interaction between extrahs was set at 1.00 and relative levels were determined. for other hepatocyte culture systems (3 to 12% with hormonally defined media [21] and 8% with dimethyl sulfoxide [11] (12,24). While a laminin receptor has not yet been identified in hepatocytes, the cell-binding activity of laminin may be related to its ability to modulate gene expression.…”

mentioning

confidence: 99%

Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins.

Caron¹

1990

Mol. Cell. Biol.

156

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Transcriptional activity of the albumin gene was induced in primary cultures of hepatocytes by adding dilute concentrations of basement membrane-like proteins derived from the EHS mouse sarcoma tumor to established type I collagen cultures. By immunofluorescence microscopy with antialbumin antibody, the population of cells responded uniformly to dilute EHS. Of the three major components of EHS, purified laminin was as effective as unfractionated EHS at inducing an increase in albumin mRNA levels and albumin secretion; type IV collagen and heparan sulfate proteoglycan were ineffective.Studies of cells in culture suggest that the extracellular matrix is involved in determining tissue-specific expression. By utilizing different culture substrata, matrices have been identified which support differentiated function in a variety of cell types (8,15,16,18) including epithelial cells (2,10,17,20,23). In primary cultures of hepatocytes, albumin synthesis and secretion is maintained for several weeks when cells are plated on a basement membrane-like matrix derived from the Engelbreth-Holm-Swarm mouse sarcoma tumor (EHS gel; described in reference 19) (4). Conversely, hepatocytes plated on a thin layer of type I collagen (TIC) exhibit a rapid loss of albumin production within 48 to 96 h. These studies have been confirmed and extended by , who showed that albumin mRNA levels parallel albumin secretion.I undertook this study to examine mechanisms by which the extracellular matrix regulates albumin gene expression. However, in addition to differences in albumin production, hepatocytes cultured on TIC and EHS gel exhibit marked differences in cell shape (3, 4) and ultrastructure (4,22). In an attempt to simplify this experimental system, I found that EHS can induce albumin expression in hepatocytes cultured on TIC. The approach involves plating hepatocytes onto TIC, allowing albumin production to decay, and then inducing expression by the addition of dilute concentrations of EHS matrix material. Addition of dilute EHS caused little perturbation to cell shape and ultrastructure while inducing a marked increase in albumin gene transcription, albumin mRNA levels, and albumin secretion. Analysis of individual components of EHS suggests that laminin is the effective agent.Induction of albumin expression by dilute EHS. Hepatocytes isolated to >95% purity from livers of adult male Sprague-Dawley rats were plated onto TIC (20 ,ug/35-mm plate) at a density of approximately 2 x 106 cells per 35-mm platein modified medium 199 with glucose, insulin, corticosterone, penicillin G, and 5% calf serum (5). The medium was changed at 4 and 24 h and daily thereafter. At 72 h after plating, when albumin secretion was greatly reduced, the medium was replaced with that containing dilute EHS (500 ,ug/ml). Within a few hours, a thin gel was visible on top of the cells. Dilute EHS medium was replaced after 24 h with normal medium (no EHS proteins), and cultures were continued with changes (normal medium) at 24-h intervals.Initial attempts to measure tr...

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Substrate adhesion of rat hepatocytes: a comparison of laminin and fibronectin as attachment proteins.

Cited by 155 publications

References 21 publications

Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin.

Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin.

Role of insulin in lipoprotein secretion by cultured rat hepatocytes.

Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins.

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