Fibronectin Gene Transcription Research Articles

Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities

Alternative splicing of the fibronectin (FN) gene transcript provides an efficient mechanism for generating functionally appropriate forms of this adhesive glycoprotein in situ. Cellular FNs that include the EIIIA and/or EIIIB FN-III segments are prominently expressed during embryogenesis, wound healing, tumor progression, and inflammation. However, the roles of this domain in altering overall FN protein structure and regulating cellular function remain unclear. We previously reported that two integrins, alpha9beta1 and alpha4beta1, ligate the EIIIA segment ( Liao, Y. F., Gotwals, P. J., Koteliansky, V. E., Sheppard, D., and Van De Water, L. (2002) J. Biol. Chem. 277, 14467-14474 ) and that the epitopes for function-blocking monoclonal antibodies lie within the C-C' loop of EIIIA ( Liao, Y. F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884 ). We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on these mutant EIIIAs. We find that the Asp(41) and Gly(42) residues within the C-C' loop of EIIIA are necessary for integrin alpha9beta1 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C' loop encode sufficient information to completely inhibit alpha9beta1-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in alpha9beta1-expressing cells accompanied by Cdc42 activation. Our data provide a cellular activity for the EIIIA segment, evidence for conformational lability, and peptide sequences for probing EIIIA functions in vitro and in vivo.

Activation of Tissue Remodeling Precedes Obliterative Bronchiolitis in Lung Transplant Recipients

Obliterative bronchiolitis (OB) and Bronchiolitis Obliterans Syndrome (BOS) are frequent complications in the lung transplant recipient, and are the leading cause of mortality after transplantation. The mechanisms responsible for OB remain elusive, but inflammatory and tissue remodeling responses are implicated. We hypothesized that alterations in markers of tissue remodeling in BALF of lung transplant recipients could predict development of OB. To test this, we identified 13 lung transplant recipients who developed both BOS and histologic OB (OB group) at median post-operative day (POD) 485 (range 73-2070). Bronchoalveolar lavage fluid (BALF) was obtained at median POD 387 (range 45-2205), which preceded the onset of OB and BOS by a median of 140 days (range 60-365). As a control, BALF was also obtained from a group of 21 stable recipients without OB (non-OB group) at median POD 335 (range 270-395). BALF was examined for gelatinolytic activity, fibronectin gene transcription, and transforming growth factor-beta1 (TGF-beta1) expression. Gelatin zymography of BALF from the OB group showed increased matrix metalloproteinase-9 (MMP-9) activity over that of the non-OB group (p < 0.005). Similarly, BALF from the OB group induced greater fibronectin expression in fibroblasts compared to the non-OB group (p < 0.03). The induction of fibronectin also correlated with the amount of TGF-beta1 protein in BALF (r = 0.71) from the OB group. We conclude that activation of tissue remodeling precedes the onset of OB, and analysis of gelatinolytic and/or fibronectin-inducing activity in BALF can serve as an early, pre-clinical marker for OB.

Increased Fibronectin Expression in Lung in the Setting of Chronic Alcohol Abuse

The incidence and severity of the acute respiratory distress syndrome (ARDS) is increased in individuals who abuse alcohol. One possible mechanism by which alcohol increases susceptibility to acute lung injury is through alterations in alveolar macrophage function and induction of tissue remodeling activity. Our objective was to determine whether alcohol abuse, independent of other comorbidities, alters fibronectin and metalloproteinase gene expression in alveolar macrophages and in epithelial lining fluid (ELF) of the lung. Otherwise healthy subjects with alcohol abuse (n=21) and smoking-matched controls (n=17) underwent bronchoalveolar lavage. Alveolar macrophage fibronectin and matrix metalloproteinase (MMP) mRNA expression were measured via reverse transcription-polymerase chain reaction. The supernatant from cultured alveolar macrophages and lung ELF were tested for their ability to induce fibronectin and MMP-9 gene transcription in cell-based assays. Alveolar macrophages from subjects with alcohol abuse demonstrated increased fibronectin mRNA expression (p<0.001), and their ELF also elicited more fibronectin gene transcription in lung fibroblasts compared with controls (p<0.001). In contrast, alveolar macrophages from subjects with alcohol abuse had decreased MMP-9 and MMP-2 mRNA expression (p<0.03 and p<0.005, respectively). Similarly, the supernatant (p<0.001) and ELF (p<0.01) from these subjects induced less MMP-9 gene transcription in THP-1 cells. Alcohol abuse is associated with increased fibronectin mRNA expression in alveolar macrophages and increased fibronectin-inducing activity in the ELF. This appears to be a specific effect as other tissue remodeling genes, such as MMPs, were not equally affected. These findings suggest activation of tissue remodeling that may contribute to the increased susceptibility for the ARDS observed in alcoholism.

Adenosine induces fibronectin expression in lung epithelial cells: implications for airway remodeling

Adenosine is an extracellular nucleoside that is elevated in tissues during hypoxia and ischemia reperfusion and has been implicated in asthma and other lung disorders. There, adenosine is considered an important modulator of physiological functions and inflammation, but its effects on matrix expression and turnover during tissue remodeling are unknown. We examined the effects of adenosine on lung epithelial cells with particular attention to the expression of fibronectin, a matrix glycoprotein highly expressed in injured tissues that has been implicated in wound healing. In A549 lung epithelial cells, we found that adenosine induced expression of fibronectin mRNA and protein in a dose- and time-dependent manner and found that the stimulatory effect of adenosine was inhibited by specific adenosine receptor antagonists. Adenosine stimulation was associated with increased levels of intracellular cAMP and with phosphorylation and DNA binding of the cAMP response element binding protein (CREB), known for its ability to stimulate fibronectin gene transcription. To confirm the latter, A549 cells were transfected with a DNA construct containing the human fibronectin promoter connected to a luciferase reporter gene. Adenosine stimulated transcription of the gene, and this effect was blocked by inhibitors of protein kinase activation. Finally, we tested primary lung fibroblasts and primary alveolar epithelial type II cells and found increased fibronectin expression in response to adenosine. Overall, our observations suggest that adenosine might modulate tissue remodeling by stimulating fibronectin expression in lung epithelial cells through induction of purinergic receptor-mediated signals that target CREB phosphorylation and stimulate fibronectin gene transcription.

Nicotine and fibronectin expression in lung fibroblasts: implications for tobacco-related lung tissue remodeling.

Tobacco-related lung diseases are associated with alterations in tissue remodeling and are characterized by increased matrix deposition. Among the matrix molecules found to be highly expressed in tobacco-related lung diseases is fibronectin, a cell adhesive glycoprotein implicated in tissue injury and repair. We hypothesize that nicotine, a component of tobacco, stimulates the expression of fibronectin in lung fibroblasts via the activation of intracellular signals that lead to increased fibronectin gene transcription. In support of this, we found that nicotine stimulated the expression of fibronectin in lung fibroblasts and that its stimulatory effect was associated with activation of protein kinase C and mitogen-activated protein kinases, increased levels of intracellular cAMP, and phosphorylation and DNA binding of the transcription factor CREB. Increased transcription of the gene was dependent on cAMP-response elements (CREs) present on the 5' end of its gene promoter. The stimulatory effect of nicotine on fibronectin expression was abolished by alpha-bungarotoxin, an inhibitor of alpha7 nicotinic acetylcholine receptors (alpha7 AChRs). Of note, nicotine increased the expression of alpha7 nAChRs on fibroblasts. Our data suggest that nicotine induces lung fibroblasts to produce fibronectin by stimulating alpha7 nAChR-dependent signals that regulate the transcription of the fibronectin gene.

The EIIIA Segment of Fibronectin Is a Ligand for Integrins α9β1 and α4β1Providing a Novel Mechanism for Regulating Cell Adhesion by Alternative Splicing

Alternative splicing of the fibronectin gene transcript gives rise to forms that include the EIIIA (or ED-A) segment. EIIIA-containing fibronectins are prominently expressed during embryogenesis and wound healing and appear to mediate changes in cell adhesion and gene expression. Nonetheless, integrins that bind the EIIIA segment have not been identified. We previously mapped the epitope for two function-blocking monoclonal antibodies to the C-C' loop region of the EIIIA segment (Liao, Y.-F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884). The sequence of this epitope ((39)PEDGIHELFP(48)) resembles the sequence within tenascin-C to which the integrin alpha(9)beta(1) binds. We now report that either integrin alpha(9)beta(1) or alpha(4)beta(1) can mediate cell adhesion to the EIIIA segment. Moreover, this interaction is blocked both by epitope-mapped EIIIA antibodies as well as by the respective anti-integrins. Deletion mutants of the EIIIA segment that include the C-C' loop and flanking sequence bind cells expressing either alpha(9)beta(1) or alpha(4)beta(1). Adhesion of alpha(4)beta(1)-containing MOLT-3 cells to the EIIIA segment stimulates phosphorylation of p44/42 MAP kinase. Our observation that two integrins bind the EIIIA segment establishes a novel mechanism by which cell adhesion to fibronectin is regulated by alternative splicing.

Regulation of serum-induced fibronectin expression by protein kinases, cytoskeletal integrity, and CREB

Lung injury, characterized by the flooding of interstitial and alveolar spaces with serum proteins, induces the expression of fibronectin (FN). This cell-adhesive extracellular matrix (ECM) glycoprotein is believed to modulate inflammation and wound repair. Murine NIH/3T3 fibroblasts transfected with a 1.2-kb human FN promoter-reporter gene were studied to gain insight into the mechanisms involved in the induction of FN by serum. Transcription of the FN gene, followed by FN protein production, was enhanced by 10% fetal bovine serum. This effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinases. ECMs typically found in injured tissues (i.e., type I collagen, fibrin, and FN) had no effect. Conversely, disruption of actin microfilaments inhibited, whereas disruption of microtubular assembly enhanced, the serum-induced FN response. The stimulatory effects of serum and microtubular disruption on FN gene transcription were related to increased DNA binding of the transcription factor cAMP response element binding protein. The data suggest that regulation of serum-induced FN expression in fibroblasts is dependent on protein kinases and on cytoskeletal integrity.

Fibronectin expression in proximal tubules from ischemic rat kidneys without reperfusion.

The expression of fibronectin (FN), one of the extracellular matrix proteins, was studied in isolated renal proximal tubules in a in vivo rat model of unilateral renal ischemia without reperfusion. FN is involved in cell-extracellular matrix interactions and defective cell-extracellular matrix interactions have been hypothesized to contribute to ischemic renal failure. The expression of FN was investigated by reverse transcription-polymerase chain reaction (RT-PCR), Elisa and Western blot. Isolated proximal tubules from control and post-ischemic rat kidneys were used. ATP, intracellular calcium content, and alkaline phosphatase were also measured to describe the effects associated to 40 min of ischemia. Control tubules expressed FN. Forty minutes of ischemia promoted diminished ATP levels and phosphatase alkaline activity, and increased intracellular calcium in isolated proximal tubules. An increased abundance of FN was observed by ischemic tubules as compared with control tubules. To determine quantitatively the value of FN content, ELISA method was performed. The ischemic tubules also expressed higher amount of FN mRNA. Three amplification products were obtained from both ischemic and control proximal tubular cDNA. The relative amounts of each of the obtained products were the same, strongly suggesting that the augmentation of the FN gene transcription during ischemia is not associated to a modification in the splicing pattern. Moreover, this expression is increased after 40 min of ischemia, not followed by reperfusion.

The Wnt/Wg signal transducer beta-catenin controls fibronectin expression.

beta-Catenin stabilizes the cadherin cell adhesion complex but, as a component of the Wnt/Wg signaling pathway, also controls gene expression by forming a heterodimer with a transcription factor of the LEF-TCF family. We demonstrate that the substrate adhesion molecule fibronectin is a direct target of Wnt/Wg signaling. Nuclear depletion of beta-catenin following cadherin transfection in Xenopus fibroblasts resulted in downregulation of fibronectin expression which was restored by activating the Wnt/Wg signaling cascade via LiCl treatment or transfection of either Xwnt-8 or beta-catenin. We isolated the Xenopus fibronectin gene (FN) promoter and found four putative LEF-TCF binding sites. By comparing the activities of different fibronectin gene reporter constructs in fibroblasts and cadherin transfectants, the LEF-TCF site at position -368 was identified as a Wnt/Wg response element. LEF-1-related proteins were found in nuclei of the fibroblasts but were absent in a kidney epithelial cell line. Consistent with the lack of these transcription factors, the FN promoter was silent in the epithelial cells but was activated upon transfection of LEF-1. Wild-type Xenopus Tcf-3 (XTcf-3) was unable to activate FN promoter reporter constructs, while a mutant lacking the groucho binding region behaved like LEF-1. In contrast to XTcf-3, LEF-1 does not interact with groucho proteins, which turn TCFs into activators or repressors (J. Roose, M. Molenaar, J. Hurenkamp, J. Peterson, H. Brantjes, P. Moerer, M. van de Wetering, O. Destreé, and H. Clevers, Nature 395:608-612, 1998). Together these data provide evidence that expressing LEF-1 enables fibroblasts, in contrast to epithelial cells, to respond to the Wnt/Wg signal via beta-catenin in stimulating fibronectin gene transcription. Our findings further promote the idea that due to its dual function, beta-catenin regulates the balance between cell-cell and cell-substrate adhesion.

Identification of Two Amino Acids within the EIIIA (ED-A) Segment of Fibronectin Constituting the Epitope for Two Function-blocking Monoclonal Antibodies

Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell alpha-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-beta-mediated smooth muscle cell alpha-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2). Mutations of pairs of amino acids to alanine have little effect, except for either (Val34Thr35) or (Tyr36Ser37), which are located in a beta strand upstream from His44. For these double mutants, the binding to all three monoclonal antibodies is markedly reduced. By contrast, single mutants at Thr35, Tyr36, or Ser37 retain full activity, suggesting that the epitope for these antibodies is determined in part by conformation. Alanine-scanning mutagenesis of rat EIIIA demonstrates the importance of Ile43 and His44 for binding. Mutation of frog EIIIA (normally Val43Lys44) to rat (Ile43His44) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile43 and His44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.

Expression of fibronectin mRNA splice variants by rabbit lung in vivo and by alveolar type II cells in vitro.

Fibronectin (FN) is a multidomain glycoprotein with putative functions in tissue development and repair. In repair of alveolar injury, FN may promote the transition of type II epithelial cells to type I epithelial cells. Alternative splicing of FN mRNA, including the EIIIA and EIIIB exons, results in protein isoforms that have cell, tissue, and developmental specificity. The present work found that FN mRNA with the EIIIA exon was in fetal, adult, and oxidant-injured lung. The EIIIB splice variant, however, was restricted to fetal lung and adult lung recovering from oxidant injury. Because alveolar type II cells in vitro express FN, we examined the splice variants in two conditions that induce FN [transforming growth factor-beta 1 (TGF-beta 1) treatment and time in culture]. TGF-beta 1 increased both EIIIA and EIIIB mRNA abundance by 10-fold. Increased EIIIA isoform immunostaining was also noted. Type II cells that spontaneously express FN at 72 h in vitro had increased EIIIA and EIIIB mRNA and increased immunostaining for EIIIA. Nuclear runoff showed induction of FN gene transcription at 72 h in vitro. Together, these data show differential FN splice variant expression in lung, with EIIIB mRNA restricted to fetal and recovering oxidant-injured lung. Furthermore, the transition of type II cells to a type I-like cell is accompanied by increased FN gene transcription and induction of both EIIIA and EIIIB mRNA.

Angiotensin II induction of fibronectin biosynthesis in cultured human mesangial cells: Association with CREB transcription factor activation

We assessed the effect of angiotensin II on fibronectin biosynthesis and transcription factor activation in adult human mesangial cells in culture. We found that 10 −5 mol/L angiotensin II tended to increase fibronectin mRNA expression within 1 hour (1.2-fold ± 0.3-fold of that in controls), with a significant increase after 4 hours (1.3-fold ± 0.1-fold of that in controls, p < 0.05) and 24 hours (1.9-fold ± 0.3-fold of that in controls, p < 0.02). In conjunction with increased fibronectin mRNA levels, angiotensin II exposure resulted in a significant elevation in immunoreactive fibronectin concentrations and the incorporation of ( 35S)-labeled methionine into fibronectin after 2 hours (224% ± 23% of controls, p < 0.05). Angiotensin II also induced mesangial cell activation of the cyclic adenosine monophosphate response element binding protein (CREB) transcription factor, a DNA binding protein known to recognize specific regulatory elements present on the fibronectin gene promoter. Using the electrophoretic mobility shift assay, we showed that angiotensin II increased mesangial cell expression of the activated form of CREB after 4 hours (1.2-fold ± 0.04-fold of that in controls, p < 0.05). To determine the importance of the CREB regulatory elements in mediating angiotensin II induction of fibronectin gene transcription, JEG-3 cells were transfected with plasmids containing fibronectin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs with (FN 510) or without (FN 122) the CREB regulatory motifs. Angiotensin II resulted in a significant increase in CAT activity in FN 510 transfectants (1. 6-fold ± 0. 2-fold of that in controls, p < 0. 05), but there was no effect of angiotensin II on FN 122 transfected cells. These data demonstrate that angiotensin II stimulates fibronectin biosynthesis in adult human mesangial cells and suggest that the process may be regulated at the transcriptional level.

G10BP, an E1A-Inducible Negative Regulator of Sp1, Represses Transcription of the Rat Fibronectin Gene

Downregulation of the fibronectin (FN) gene in a rat 3Y1 derivative cell line, XhoC, transformed by the adenovirus E1A and E1B genes seems to be caused by the induction of a negative regulator, G10BP, which binds to three G-rich sequences in the promoter (T. Nakamura, T. Nakajima, S. Tsunoda, S. Nakada, K. Oda, H. Tsurui, and A. Wada, J. Virol. 66:6436-6450, 1992). These are the G10 stretch and two GC boxes consisting of the G10 stretch with one internal C residue insertion. The recognition sequences of G10BP and Sp1 (GGGCGG) overlap in these GC boxes. To analyze the mechanism of the downregulation, G10BP was purified by DNA affinity chromatography, and its molecular mass was estimated to be about 30 kDa. The promoter was modified by substituting the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, and by replacing the Sp1 motif by the T stretch. Transcription of FN promoter-chloramphenicol acetyltransferase fusion genes carrying the base substitution in one or more of these G-rich sequences both in vivo and in vitro revealed that the base substitution in any G-rich sequence results in reduction of promoter activity, although the downstream GC box (GCd) plays a primary role. The addition of G10BP severely inhibited the activities of the FN promoters carrying the wild-type GCd in vitro, while the promoters carrying the mutant GCd were unaffected. The binding affinity of G10BP and Sp1 to each of the G-rich sequences, analyzed by gel shift assays, indicated that G10BP binds strongly to the GCd, moderately to the G10 stretch, and weakly to GCu, while Sp1 binds strongly to GCu, moderately to GCd, and weakly to the G10 stretch. Sp1 binding to GCd and the G10 stretch was inhibited by G10BP, while binding to GCu was unaffected. These results indicate that FN gene transcription is inhibited in XhoC cells primarily by exclusion of Sp1 binding to GCd by G10BP and that G10BP is a new class of Sp1 negative regulator.

Transcriptional Repression of Fibronectin Gene Expression in v-src Transformation

V-src-dependent and -independent alterations in the steady-state content, rates of synthesis, and turnover of fibronectin protein and mRNA were identified using rat fibroblasts which are temperature sensitive for p60v-src activity. Activation of p60v-src caused a fivefold reduction in the rate of fibronectin biosynthesis. The v-src-dependent decrease in fibronectin biosynthesis resulted from a similar reduction in the steady-state content of fibronectin mRNA. This change was reversible and required more than 24 h, implying an indirect effect of p60v-src on fibronectin gene expression. The rate of fibronectin mRNA turnover and pattern of alternative splicing were unchanged following p60v-src activation, indicating that these regulatory steps are insensitive to v-src transformation. A v-src-specific reduction of at least threefold was measured for the rate of fibronectin gene transcription, and gene transfer studies using fibronectin promoter-CAT reporter genes indicated that transcriptional repression occurs at the level of initiation. When p60v-src was inactive, CAT reporter genes controlled by 4.9 or 3.2 kb of the rat fibronectin promoter exhibited relatively increased CAT activity (∼twofold) compared to another CAT reporter construction controlled by only 880 bp of the fibronectin promoter. In contrast, CAT activity was relatively reduced (∼twofold) for the reporter constructions containing 4.9 or 3.2 kb of the promoter when p60v-src was active. These findings indicate that the distal portion of the fibronectin promoter contains a v-src-sensitive element(s) which mediates a decrease in the rate of fibronectin transcription initiation by negative control.

NEGATIVE REGULATION OF FIBRONECTIN GENE-TRANSCRIPTION BY EJ-HA-RAS AND C-SIS ONCOGENES IN BALB/C 3T3 CELLS

Down-regulation of fibronectin gene expression at cell surfaces by various oncogenes is an important phenotypic change of many transformed cells. Mechanisms effecting this loss of cell surface fibronectin remain poorly understood. An isogenic mouse fibroblast system, Balb/c 3T3, was used to investigate the effect of oncogene transformation on fibronectin gene transcription. Fibronectin mRNA levels were shown to decrease by >80% when 3T3 cells became stably transformed by EJ-Ha-ras or c-sis oncogenes. The mouse fibronectin gene promoter was subsequently cloned from a mouse genomic library and inserted into reporter gene constructs. Transient transfection assays with the 1.2 kb fibronectin promoter demonstrated that its activity was also decreased in the two transformed cell lines to a similar extent as fibronectin mRNA levels. Co-transfections into 3T3 cells with both the promoter construct and either Ha-ras or c-sis expression constructs provided further support for negative regulation of fibronectin gene transcription by oncogene expression. Potential mechanisms for such transcriptional regulation are proposed based on deletion analysis of the fibronectin promoter. These data suggest that down-regulation of fibronectin gene transcription can contribute directly to the loss of cell surface fibronectin in transformed murine fibroblasts.

Expression of fibronectin during rat fetal and postnatal development: an in situ hybridisation and immunohistochemical study.

Fibronectin is a protein of the extracellular matrix with numerous binding sites to the other elements of the matrix and to the cells. The aim of this study was to determine the relative importance of fibronectin isoform expression (FN-EIIIA, FN-EIIIB) during fetal and postnatal development of the rat heart. In situ hybridisation and immunolabelling approaches were used to describe the cellular synthesis of fibronectin and its distribution throughout the rat heart from 11 d postconception until adulthood. The distribution of fibronectin was compared to that of laminin and of alpha type III procollagen. The accumulation and pattern of distribution of the major fibronectin mRNA isoforms were identical, that is, there was a progressive decrease in their accumulation as a function of time after 11 d postconception, resulting in a complete absence in the adult. The distribution of fibronectin and procollagen type III mRNAs were, however, quite distinct. At the protein level the time course of synthesis and secretion of the locally synthesised fibronectin (c-FN) did not follow fibronectin mRNA expression, the accumulation of the protein being rather poor, except just before birth, where it was found mainly in the coronary vessels. During the development of the fetal rat heart fibronectin gene transcription is active and progressively decreases with age, whereas the translation of the mRNAs into their corresponding proteins is always relatively poor. If fibronectin is involved in fetal and postnatal morphogenesis of the rat myocardium, it is the plasma form (p-FN) that is most probably involved in the process of growth and differentiation of the rat heart.

Forskolin induces the reorganization of extracellular matrix fibronectin and cytoarchitecture in 3T3-F442A adipocytes: its effect on fibronectin gene expression

We studied the effect of forskolin on fibronectin and actin gene expression in 3T3-F442A adipogenic cell line. The structural organizations of extracellular matrix fibronectin and actin cytoskeleton were investigated in parallel. Immunofluorescence experiments showed that preadipocytes treated for 48 h with 10 μM forskolin exhibited an intensified network of both actin and fibronectin when compared to control. A similar picture was obtained with adipocytes given long-term exposure to forskolin. As determined by Western analysis, fibronectin protein levels were increased by 50–75% over control, both in preadipocytes and adipocytes. A parallel increase of fibronectin mRNA content was observed in forskolin-treated cells. In contrast, forskolin treatment of preadipocytes and adipocytes did not elicit any change in the steady-state level of either actin mRNA or protein. Nuclear run-on experiments showed that forskolin increased the fibronectin gene transcription rate but left that of the actin gene in adipocytes unchanged. These findings suggest the reorganization of the actin network in forskolin-treated adipocytes to be a consequence of fibronectin-enhanced biosynthesis and reorganization.

ACETALDEHYDE INDUCES C-FOS AND C-JUN PROTO-ONCOGENES IN FAT-STORING CELL CULTURES THROUGH PROTEIN KINASE C ACTIVATION

Hepatic fibrosis is an important morphological feature of alcohol-induced liver injury. We previously reported that acetaldehyde stimulates collagen I and fibronectin gene transcription in rat fat-storing cell (FSC) culture. We here evaluated whether acetaldehyde increases Col I and FN gene transcription through the induction of c-fos and c-jun proto-oncogenes and studied the possible role played by protein kinase C (PKC) and c-AMP. FSCs, isolated from rat liver on a Nycodenz density gradient, were exposed to acetaldehyde for 1/2, 1, 3, 6, 12, 24 hr and for 10, 20, 30, 45, 60, 90 min in the experiments for jun and fos expression, respectively. Acetaldehyde produced a rapid and transient induction of fos mRNA (undetectable at t = 0, peak at t = 45 and still evident at t = 90). Jun mRNA was weakly expressed in unstimulated FSCs; acetaldehyde induced a prolonged activation of jun expression up to 24 hr with a peak at 3 hr. To study the role of PKC were repeated the experiments in the presence of Staurosporine and H-7. These inhibitors of PKC activity blocked the stimulatory effect of acetaldehyde on fos and jun mRNA expression. Furthermore, they abolished the stimulatory effect of acetaldehyde on collagen I and fibronectin gene expression by FSCs. Acetaldehyde increased the cell membrane PKC activity in FSC cultures in a dose-dependent way. Intracellular cAMP levels were not significantly modified by acetaldehyde in the first 30 min of incubation. We conclude that acetaldehyde increases procollagen I and fibronectin gene transcription in FSCs, possibly through c-fos and c-jun expression, and that PKC may play a regulatory role in this chain of events.

Regulation of Alternative Splicing of the Fibronectin IIICS Domain by Cytokines

The primary fibronectin gene transcript is alternatively spliced in three regions, designated EIIIA, EIIIB and IIICS. While the functions of the EIIIA and EIIIB domains are still to be established, either end of the IIICS domain contains the cell-binding sites CS1 and CS5 recognised by the integrin VLA-4 which is present on mononuclear cells. We have determined the effects of three cytokines upon fibronectin mRNA and IIICS splice variants in four cultured human cell lines using Northern hybridisation and PCR. In the cell line CAKI-2, interleukin 1β, Platelet-derived growth factor BB and transforming growth factor β1 all increased mRNA splice variants where the CS1 and CS5 regions were removed. This cytokine-induced change would lead to a decrease in the CS1 and CS5 cell binding sites within the extracellular matrix. All the changes in spliceosome function increase use of downstream 3′ splice acceptor sites and may represent use of a single common pathway by three different cytokines.

Acetaldehyde-protein adducts, but not lactate and pyruvate, stimulate gene transcription of collagen and fibronectin in hepatic fat-storing cells

Hepatic fibrosis is an important morphological feature of alcohol-induced liver injury. We previously reported that acetaldehyde, but not ethanol can stimulate type I collagen and fibronectin synthesis in cultures of rat fat-storing cells (FSC) by increasing transcription of the specific genes. The effect of lactate and pyruvate was studied on collagen I, III, fibronectin accumulation by cultured rat FSCs and it was investigated whether acetaldehyde could increase procollagen I and fibronectin gene transcription through the formation of protein adducts. Lactate and pyruvate (5, 15 and 25 mmol/l) did not significantly affect collagen I, III and fibronectin production by cultured FSCs. Pyridoxal-phosphate and p-hydroxymecuribenzoate (inhibitors of acetaldehyde-protein adduct formation) blocked the stimulatory effect of acetaldehyde on procollagen I and fibronectin gene transcription. These data suggest that ethanol may act as a liver fibrogenic factor through acetaldehyde, its immediate metabolite, whereas lactate does not seem to play a role. Acetaldehyde might stimulate gene transcription of extracellular matrix components by liver FSCs through the formation of adducts with proteins.