Fibronectin Gene Transcription Research Articles

Fibronectin Gene Transcription Is Enhanced in Abnormal Wound Healing

Abnormal wound healing in susceptible individuals can result in the formation of keloids that have an elevated content of extracellular matrix material compared to normal scars. Keloid-derived fibroblasts exhibit as much as a four-fold increase in the rate of fibronectin biosynthesis compared to fibroblasts from normal dermis and normal scars. Altered biosynthesis is due to an increase in the steady-state level of fibronectin mRNA, and in this investigation we have identified the level of fibronectin gene expression that is responsible for this increase. The rate of fibronectin gene transcription was found to be increased as much as threefold in keloid fibroblasts when compared to normal fibroblasts. Other possible changes that could account for the elevated level of fibronectin mRNA in keloids, such as increased copy number of the fibronectin gene or decreased turnover of fibronectin mRNA were also examined. The possibility of altered gene dosage was eliminated because chromosome content, G-banding patterns, and fibronectin gene content of keloid fibroblasts were all found to be normal. Analysis of fibronectin mRNA degradation revealed a half-life of approximately 13 h, and the residual fibronectin mRNA was observed to remain full length during this time period in both keloid and normal fibroblasts. Thus, altered degradation of fibronectin mRNA is unlikely to contribute to overproduction of fibronectin in keloids. Increased translational competence of fibronectin mRNA in keloids was also eliminated as a contributing factor because fibronectin mRNA remaining after one half-life were equally available for translation in both cell types. Although stimulation of transcription may not entirely account for the increase in fibronectin biosynthesis in keloids, this mechanism is best able to account for the majority of the change.

Induction of E1A-responsive negative factors for transcription of the fibronectin gene in adenovirus E1-transformed rat cells.

The level of fibronectin (FN) gene expression is very high in resting rat 3Y1 cells but greatly decreased in adenovirus E1-transformed cells. To study the mechanism of this down-regulation, nuclear factors binding to the 5'-flanking region of the FN gene were analyzed by gel retardation assay and DNase I footprinting. Nuclear factors that were present in the transformed cells but nearly absent in resting 3Y1 cells interacted with multiple sites of the promoter region. Oligonucleotide competition with the FN promoter-chloramphenicol acetyltransferase (CAT) reporter constructs (pFCAT) for these factors in the transformed cells indicated that all of them had a negative effect on FN gene expression. Of them, a factor(s) (G10BP) binding to the G10 stretch from positions -239 to -230 and to two GC boxes consisting of the G10 stretch with one internal C residue insertion from positions -105 to -95 and -54 to -44 had the strongest repressive activity. Introduction of substitutive mutations into these G-rich sequences resulted in the increase in CAT activity of pFCAT in the transformed cells. The recognition sequences of G10BP and Sp1 overlap in two GC boxes. G10BP has stronger affinity for heparin and GC boxes than does Sp1, suggesting that G10BP may repress FN gene transcription by displacing Sp1.

E1A-responsive elements for repression of rat fibronectin gene transcription.

The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of E1A expression. To study the mechanism of this E1A-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with E1A expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary E1A-responsive elements for repression of the FN gene. Two GC boxes also contain a G10 stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Sp1 motif GGGCGG. Substitution of the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, completely abolished the E1A sensitivity of the promoter. Analysis of the E1A domains by using various E1A deletion mutants indicated that the domain for binding to the retinoblastoma susceptibility gene product (RB) is essential for efficient repression. These results suggest that the gene encoding a negative factor(s) binding to the three G-rich sequences in the FN promoter is repressed by RB in resting 3Y1 cells and derepressed by expression of E1A.

E1A-responsive elements for repression of rat fibronectin gene transcription.

The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of E1A expression. To study the mechanism of this E1A-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with E1A expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary E1A-responsive elements for repression of the FN gene. Two GC boxes also contain a G10 stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Sp1 motif GGGCGG. Substitution of the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, completely abolished the E1A sensitivity of the promoter. Analysis of the E1A domains by using various E1A deletion mutants indicated that the domain for binding to the retinoblastoma susceptibility gene product (RB) is essential for efficient repression. These results suggest that the gene encoding a negative factor(s) binding to the three G-rich sequences in the FN promoter is repressed by RB in resting 3Y1 cells and derepressed by expression of E1A.

Acetaldehyde increases procollagen type I and fibronectin gene transcription in cultured rat fat-storing cells through a protein synthesis-dependent mechanism, ,

We previously reported that acetaldehyde increases the production of type I collagen in cultured rat fat-storing cells. We studied the regulation of this effect by determining the expression of procollagen type I, fibronectin and transforming growth factor-beta 1 messenger RNAs in passage 1 and 2 cultures of fat-storing cells exposed to acetaldehyde for up to 24 hr. By 6 hr, acetaldehyde increased the steady-state levels of alpha 1 procollagen type I messenger RNA 3.2-fold and of fibronectin messenger RNA 2.8-fold above control values. At 24 hr, messenger RNA levels remained elevated. In contrast, transforming growth factor-beta 1 messenger RNA steady-state levels remained unaltered by 6 hr, but increased 1.5-fold by 24 hr. Cycloheximide (0.3 mmol/L) completely inhibited the acetaldehyde effect when added at zero time but was less effective when added at 15 min. The effect of acetaldehyde was not modified when cells were cultured in the presence of the acetaldehyde dehydrogenase inhibitor cyanamide (100 mumol/L). Fat-storing cells were also cultured in the presence of lactate (5, 15 and 25 mmol/L) for 6 hr. At none of these concentrations was any effect seen on either alpha 1(I) procollagen or fibronectin messenger RNAs. In the presence of methylene blue, a scavenger of reducing equivalents, the effect of acetaldehyde on alpha 1(I) procollagen and fibronectin gene expression was totally inhibited. Transcription run-on assay showed that acetaldehyde increased both procollagen type I and fibronectin transcriptional activity threefold and 2.5-fold, respectively. We conclude that acetaldehyde increases alpha 1(I) procollagen and fibronectin gene expression through enhanced transcription by a mechanism dependent on newly synthesized proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde increases procollagen type I and fibronectin gene transcription in cultured rat fat-storing cells through a protein synthesis-dependent mechanism

We previously reported that acetaldehyde increases the production of type I collagen in cultured rat fat-storing cells. We studied the regulation of this effect by determining the expression of procollagen type I, fibronectin and transforming growth factor-beta 1 messenger RNAs in passage 1 and 2 cultures of fat-storing cells exposed to acetaldehyde for up to 24 hr. By 6 hr, acetaldehyde increased the steady-state levels of alpha 1 procollagen type I messenger RNA 3.2-fold and of fibronectin messenger RNA 2.8-fold above control values. At 24 hr, messenger RNA levels remained elevated. In contrast, transforming growth factor-beta 1 messenger RNA steady-state levels remained unaltered by 6 hr, but increased 1.5-fold by 24 hr. Cycloheximide (0.3 mmol/L) completely inhibited the acetaldehyde effect when added at zero time but was less effective when added at 15 min. The effect of acetaldehyde was not modified when cells were cultured in the presence of the acetaldehyde dehydrogenase inhibitor cyanamide (100 mumol/L). Fat-storing cells were also cultured in the presence of lactate (5, 15 and 25 mmol/L) for 6 hr. At none of these concentrations was any effect seen on either alpha 1(I) procollagen or fibronectin messenger RNAs. In the presence of methylene blue, a scavenger of reducing equivalents, the effect of acetaldehyde on alpha 1(I) procollagen and fibronectin gene expression was totally inhibited. Transcription run-on assay showed that acetaldehyde increased both procollagen type I and fibronectin transcriptional activity threefold and 2.5-fold, respectively. We conclude that acetaldehyde increases alpha 1(I) procollagen and fibronectin gene expression through enhanced transcription by a mechanism dependent on newly synthesized proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor beta, and cAMP in human cell lines.

The regulation of fibronectin (FN) biosynthesis by dexamethasone (a synthetic glucocorticoid), forskolin (an activator of adenylate cyclase), and transforming growth factor beta (TGF-beta) was examined in six human cell lines. Dexamethasone treatment produced the largest increase in FN biosynthesis in the fibrosarcoma cell line, HT-1080 (approximately 45-fold). This seems to result from a dexamethasone-mediated increase in FN mRNA stability which increases the message half-life from approximately 11 to 26 h. The relative instability of FN mRNA in the fibrosarcoma (t1/2 11 h) compared to normal fibroblasts (70 h) appears to result from the particular transformed phenotype of the HT-1080 cells. Forskolin and TGF-beta increase the rate of FN gene transcription in most of the cell lines. These effects (four- to six-fold) occur rapidly and do not require protein synthesis in the responsive cell lines which include normal fibroblasts. However, in the fibrosarcoma (HT-1080), a surprisingly large induction (20-30-fold) is observed and this induction is different from that in the normal fibroblasts and the other cell lines in that both protein synthesis and a lag period are required. Synergism is seen with dexamethasone and either forskolin or TGF-beta in HT-1080 cells increasing the rate of FN biosynthesis approximately 200-fold to a level similar to normal fibroblasts. This seems to result from a combination of FN mRNA stabilization (dexamethasone) and increased transcription (forskolin and TGF-beta).

Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells.

A cDNA library, prepared from poly(A)+ RNA isolated from quiescent AKR-2B cells 4 hr after stimulation with epidermal growth factor in the presence of cycloheximide, was screened to identify RNA transcripts whose abundance is specifically increased as a primary response to growth stimulation. Approximately 40% of the inducible clones detected by this procedure corresponded to either cytoskeletal beta- or gamma-actin genes. One nonactin clone, designated c99, was found to be derived from an 8.5-kilobase RNA whose abundance began to increase as early as 30 min after stimulation. DNA sequencing established the identity of this RNA as fibronectin. Several additional mitogens were then tested and found to efficiently induce fibronectin mRNA. These included fetal calf serum, platelet-derived growth factor, and transforming growth factor type beta. For at least one inducer, fetal calf serum, the increase in mRNA was preceded by an increase in fibronectin gene transcription. This increase was rapid, reaching maximal levels within 10 min, and was accompanied by near-coordinate increases in both c-fos and beta-actin transcription. These results indicate that fibronectin is a member of a class of "early-response" genes, typified by c-fos and including beta-actin, whose rapid expression may be important in mediating cellular responses to peptide growth factors.

Multiple sites of alternative splicing of the rat fibronectin gene transcript.

We describe analyses of the structure and expression of the rat fibronectin gene with particular attention to the 40-kb stretch from the center of the gene which encodes 17 type-III repeating units. Each repeat is precisely separated from its neighbors by introns and most are encoded by pairs of exons. Three repeats are encoded precisely by single exons and two of these (EIIIA and EIIIB) are alternatively spliced in a cell type-specific fashion. A third site of alternative splicing (EIIIB) reported here is similar in expression to the previously described EIIIA segment. Both are excluded from mRNA in liver cells and are, therefore, absent from plasma fibronectin. These two alternative splices, plus a third one (V) reported previously, can occur in all possible combinations giving 12 fibronectin mRNAs from a single gene. These splicing variations account for most but not all of the known fibronectin subunit variants. We report investigations designed to detect other regions of alternative splicing. We also show that the pattern of alternative splicing is somewhat altered on oncogenic transformation.

Donor and acceptor splice signals within an exon of the human fibronectin gene: a new type of differential splicing

We have sequenced that area of a human fibronectin gene clone which codes for a connecting strand separating the last two areas of the type III homology. The gene has a complex exon with two ‘AG’ acceptor sites. One of these can be used (exon subdivision). In addition 93 basepairs inside the exon are sometimes spliced out as an intron. This is the third differential splicing found in the fibronectin gene transcript and it represents a new type of differential splicing.