Biomedical subjects

T M Birkenmeier

Publications and source records attributed to T M Birkenmeier.

10 recordsLinked to original sources

PubMed1996

Integrin regulation by endogenous expression of 18-kDa fibroblast growth factor-2.

The three high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) have a distinct intracellular localization and differentially affect cell mobility and growth compared with the fourth 18-kDa form. To characterize further the effects of the 18-kDa and HMW forms of FGF-2, we have examined their ability to modulate integrin expression. Transfected NIH 3T3 cells expressing only 18-kDa FGF-2 exhibited increased cell surface levels of alpha5beta1, whereas cells expressing only HMW FGF-2 exhibited cell surface alpha5beta1 levels similar to parental cells. When cells synthesizing 18-kDa FGF-2 were transfected with a cDNA encoding a dominant negative FGF receptor, alpha5beta1 cell surface levels decreased. Immunoprecipitation of biosynthetically labeled cells indicated that expression of 18-kDa FGF-2 increased the biosynthesis and rate of maturation of alpha5. Northern blot analysis showed that 18-kDa FGF-2 increases the level of the alpha5 subunit mRNA but does not affect beta1 subunit transcript levels. Experiments utilizing luciferase reporter gene activity revealed increased alpha5 promoter activity in cells expressing 18-kDa FGF-2 indicating that the enhanced alpha5 transcript level is due to modulation of the transcription rate. Therefore, interaction of 18-kDa FGF-2 with FGF receptors results in changes in alpha5beta1 biosynthesis and processing. In contrast, endogenous expression of HMW FGF-2 does not mediate this effect.

3T3 Cells↗

PubMed1995

Autocrine transforming growth factor beta 1 modulates the expression of integrin alpha 5 beta 1 in human colon carcinoma FET cells.

Transforming growth factor beta (TGF-beta) has been extensively studied as an exogenous agent that stimulates the expression of extracellular matrix proteins and their cell-surface integrin receptors in a variety of cell types. However, the recent demonstration of autocrine TGF-beta growth effects in a number of cell types suggests that the steady-state expression of extracellular matrix and integrin proteins and their biological activity may also be under autocrine TGF-beta control. Previously, we reported that repression of autocrine TGF-beta 1 activity by constitutive expression of a full-length TGF-beta 1 antisense cDNA led to abrogation of autocrine negative TGF-beta and, as a result, increased tumorigenicity and anchorage-independent growth of a poorly tumorigenic, well-differentiated colon carcinoma cell line designated FET (Wu, S., Theodorescu, D., Kerbel, R. S., Willson, J. K. V., Mulder, K. M., Humphrey, L. E., and Brattain, M. G. (1992) J. Cell Biol. 116, 187-196). Consequently, we have used this model system to study the effects of repression of autocrine TGF-beta 1 activity on the expression of integrin alpha 5 beta 1 and integrin alpha 5 beta 1-mediated cell adhesion to fibronectin. The expression of the integrin alpha 5 subunit was reduced in TGF-beta 1 antisense transfected FET cells at both mRNA and protein levels as determined by RNase protection assays and immunoprecipitation, respectively. Autocrine TGF-beta 1 had no effect on the transcription of integrin alpha 5 and beta 1 subunits, indicating that autocrine TGF-beta 1 may regulate integrin alpha 5 beta 1 expression at the post-transcriptional level. The diminished expression of integrin alpha 5 beta 1 on the cell surface led to the reduced adhesion of TGF-beta 1 antisense transfected cells to fibronectin. This phenomenon could be reversed by treatment with exogenous TGF-beta 1.

Cell Adhesion↗

PubMed1995

Release from quiescence stimulates the expression of integrin alpha 5 beta 1 which regulates DNA synthesis in human fibrosarcoma HT1080 cells.

We show that integrin alpha 5 subunit expression is stimulated when human fibrosarcoma HT1080 cells are released from quiescence. The alpha 5 subunit mRNA level in quiescent HT1080 cells was increased 24 hr after their release by 10% fetal bovine serum-containing medium reaching a maximum of 2.5 fold on day 2. Similar levels of induction of cell-surface alpha 5 subunit protein as well as beta 1 subunit protein were also observed. This resulted in a significant increase of cell attachment to fibronectin. The serum stimulation also increased alpha 5 subunit promoter activity by twofold which was protein synthesis independent. Subsequent deletion of alpha 5 subunit promoter DNA showed that the cis-element responsible for the activation is located between -92 bp and the transcription start site. The promoter activity was not induced until 12 hr after the release. Comparison of the effect of a serum-free medium and a 10% fetal bovine serum-supplemented medium revealed that both the DNA synthesis and alpha 5 subunit induction were independent of exogenous growth factors. The increased integrin alpha 5 beta 1 appears to function by reducing mitogenic activity since blockade of fibronectin binding to its receptor with a RGD peptide, a monoclonal anti-fibronectin antibody, or a monoclonal anti-alpha 5 subunit antibody during the release from quiescence significantly stimulated DNA synthesis. On the other hand, stable overexpression of the alpha 5 subunit resulted in decreased DNA synthesis.

Cell Adhesion↗

PubMed1991

Identification of a protein that interacts with the nuclear factor-1 (NF-1) binding site in cells that do not express NF-1: comparison to NF-1, cellular distribution, and effect on transcription.

We examined expression of nuclear factor-1 (NF-1) in different cell lines. Expression was low or undetectable in T and B lymphocyte cell lines, whereas fibroblasts and other adherent cell lines generally had a relatively high level of NF-1 mRNA. In cell lines that did not express NF-1, gel retardation assays, nevertheless, indicated complexes between a protein or proteins and the NF-1 site. These complexes were less abundant than those formed with NF-1, they migrated more slowly, and they appeared as single species instead of the multiple species observed with NF-1. NF-1 site-binding proteins were compared in the fibrosarcoma cell line HT-1080 (expressed the highest level of NF-1 in our study) and the B cell line Raji (does not express NF-1). UV-crosslinking studies indicated that the NF-1 site-binding proteins in both cell lines were similar in size. Proteolytic clipping band shift assays suggested that the Raji protein and NF-1 share structural similarity in their DNA binding domains, but are distinct proteins. The NF-1 site mediated transcriptional stimulation in cell lines where NF-1 is expressed; however, this element did not affect transcription in cell lines that do not express NF-1, suggesting that the NF-1 site-binding protein in these cells is functionally distinct from NF-1.

3T3 Cells↗

PubMed1991

The alpha 5 beta 1 fibronectin receptor. Characterization of the alpha 5 gene promoter.

A genomic clone containing the gene for the alpha 5 subunit of the human alpha 5 beta 1 integrin complex was isolated by screening a human genomic library with the previously described alpha 5 cDNA (Argraves, W. S., Suzuki, S., Arai, H., Thompson, K., Pierschbacher, M. D., and Ruoslahti, E. (1987) J. Cell Biol 105, 1183-1190). The alpha 5 gene 5'-flanking region lacks both TATA and CCAAT boxes, and it is located in a CpG island. This region was an active promoter in transfection assays using the HT-1080 cell line (fibrosarcoma), which expresses alpha 5, but was inactive in the Raji cell line (B cell), which does not express alpha 5. These results indicate that the alpha 5 gene 5'-flanking region acts as a promoter that exhibits the expected cell-type specificity. Deletion of alpha 5 promoter sequences from position -657 to -178 caused transcription stimulation, suggesting that a silencer is located between these sites. Successive 5' deletions from position -178 decreased promoter activity until activity was essentially eliminated on deletion to position -27. Isolation of a functional promoter for the alpha 5 gene is a first step in understanding how expression of this gene is controlled at the molecular level.

Amino Acid Sequence↗

PubMed1991

Cell surface molecules that bind fibronectin's matrix assembly domain.

The assembly of fibronectin into disulfide cross-linked extracellular matrices requires the interaction of mesenchymal cells with two distinct sites on fibronectin, the Arg-Gly-Asp cell adhesive site and an amino-terminal site contained within the first five type I homologous repeats (Quade, B. J., and McDonald, J. A. (1988) J. Biol. Chem. 263, 19602-19609). Proteolytically derived 29-kDa fragments of fibronectin (29kDa) containing these repeats bind to monolayers of cultured fibroblasts and inhibit fibronectin matrix assembly. The cell surface molecules interacting with fibronectin's 29-kDa matrix assembly domain have resisted purification using conventional methods such as affinity chromatography. Accordingly, in order to identify molecules which bind this fragment, 125I-labeled 29kDa was allowed to bind to fibroblast monolayers and chemically cross-linked to the cell surface with bis(sulfosuccinimidyl) suberate. Extraction of the cross-linked cell layer yielded radiolabeled complexes of 56, 150, and 280 kDa. Formation of these cross-linked complexes was specifically inhibited by the addition of excess unlabeled 29kDa but was unaffected by the presence of fibronectin fragments containing other type I repeats outside of the 29kDa matrix assembly domain. The cross-linked complexes were insoluble in nondenaturing detergents but soluble when denatured and reduced, suggesting that 29kDa may be cross-linked to components of the pericellular matrix. Immunoprecipitation of cross-linked cell extracts with a polyclonal antibody to fibronectin that does not recognize the amino terminus demonstrate that the 280-kDa band contains 29kDa cross-linked to fibronectin present on the cell surface. Formation of the 150-kDa complex was inhibited by EDTA, suggesting that divalent cations are required for its formation. Although the molecular mass and divalent cation requirement suggest that the 150-kDa complex may be related to an integrin, this complex was not immunoprecipitated by polyclonal antibodies generated to the alpha 5 beta 1 integrin fibronectin receptor.

Animals↗

PubMed1991

Characterization of the alpha 4 integrin gene promoter.

A cDNA for the alpha 4 chain of the alpha 4 beta 1 integrin was described previously [Takada, Y., Elices, M. J., Crouse, C. & Hemler, M. E. (1989) EMBO J. 8, 1361-1368]. Primer extension analysis indicated that alpha 4 mRNA extended well beyond the 5' end of this cDNA. To clone this 5' sequence, a primer extension cDNA library was constructed, and a cDNA extending an additional 660 base pairs was isolated. This cDNA hybridized to multiple mRNAs in both T and B lymphocytes, but no alpha 4 mRNA was found in different tissues or in adherent cell lines. A single alpha 4 gene was detected in a genomic Southern blot when hybridization was done at high stringency; however, additional bands were observed at lower stringency, indicating the presence of alpha 4-related genes. Some of the different mRNAs that hybridize to the alpha 4 cDNA may then be the products of these related genes. Analysis of the alpha 4 genomic sequence revealed a large first exon of 958 base pairs. Interestingly, translation of alpha 4 initiates from the second ATG in this exon (nucleotide + 744). The first ATG (nucleotide +21) is followed by a termination codon 21 amino acids downstream. Such upstream ATG codons have been implicated in translational control of protooncogenes. One major transcriptional start site was identified by using S1 nuclease and primer extension mapping. Consensus sequences for DNA regulatory elements were found upstream of the gene and in exon 1 and intron 1. The alpha 4 gene 5' flanking region acted as a promoter in transfection assays. Detailed characterization of the promoter should provide insight into molecular events regulating expression and tissue specificity of alpha 4.

Amino Acid Sequence↗

PubMed1991

Glycoprotein synthesis and secretion. Expression of fibronectin and its cell surface receptors.

Fibronectin (FN) is an extracellular matrix protein that acts as a substrate for cell migration and adhesion during development. Cells adhere to FN through integral membrane proteins that are members of the integrin family of adhesion molecules. The interaction between cells and FN is important in a number of biologic processes, including gastrulation, hematopoietic differentiation, neural crest cell migration, cardiac development, branching morphogenesis in lung, wound healing, tumorigenesis, and metastasis. Expression of FN and its receptors is controlled by a number of hormones and growth factors as well as by tissue-specific factors. Here, the molecular aspects of how expression of these genes is controlled are reviewed, with particular emphasis on promoter regulator elements that modulate expression.

Fibronectins↗

PubMed1989

Identification of a cyclooxygenase-related gene and its potential role in prostaglandin formation.

Regulation of cyclooxygenase expression was studied in homogenous preparations of epithelial cells isolated from sheep tracheal mucosa. Cellular capacity to generate cyclooxygenase-derived arachidonate metabolites (predominantly prostaglandin E2) increased markedly in cultured compared to freshly isolated cells. A 70 kDa cyclooxygenase protein and corresponding 2.8 kb mRNA were coordinately expressed but their levels did not increase proportionately to the increase in cyclooxygenase activity. Rehybridization of Northern blots at lower stringency revealed the presence of a new tissue-specific 4.0 kb mRNA species exhibiting increased expression during cell culture. Hybridization of the 4.0 kb mRNA with two nonoverlapping cDNA probes at only low stringency conditions suggests that it is derived from a distinct gene. Its relatedness to cyclooxygenase and its increase in parallel with enzymatic activity further suggest that the larger mRNA may encode for a cyclooxygenase.

Animals↗

PubMed1988

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts.

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.

Cell Line↗