Expression In Mouse Fibroblasts Research Articles

TGFβ1-RCN3-TGFBR1 loop facilitates pulmonary fibrosis by orchestrating fibroblast activation

BackgroundIdiopathic pulmonary fibrosis (IPF) bears high mortality due to unclear pathogenesis and limited therapeutic options. Therefore, identifying novel regulators is required to develop alternative therapeutic strategies.MethodsThe lung fibroblasts from IPF patients and Reticulocalbin 3 (RCN3) fibroblast-selective knockdown mouse model were used to determine the importance of Rcn3 in IPF; the epigenetic analysis and protein interaction assays, including BioID, were used for mechanistic studies.ResultsReticulocalbin 3 (RCN3) upregulation is associated with the fibrotic activation of lung fibroblasts from IPF patients and Rcn3 overexpression blunts the antifibrotic effects of pirfenidone and nintedanib. Moreover, repressing Rcn3 expression in mouse fibroblasts ameliorates bleomycin-induced lung fibrosis and pulmonary dysfunction in vivo. Mechanistically, RCN3 promotes fibroblast activation by maintaining persistent activation of TGFβ1 signalling via the TGFβ1-RCN3-TGFBR1 positive feedback loop, in which RCN3 upregulated by TGFβ1 exposure detains EZH2 (an epigenetic methyltransferase) in the cytoplasm through RCN3-EZH2 interaction, leading to the release of the EZH2-H3K27me3 epigenetic repression of TGFBR1 and the persistent expression of TGFBR1.ConclusionsThese findings introduce a novel regulating mechanism of TGFβ1 signalling in fibroblasts and uncover a critical role of the RCN3-mediated loop in lung fibrosis. RCN3 upregulation may cause resistance to IPF treatment and targeting RCN3 could be a novel approach to ameliorate pulmonary fibrosis.Graphical

Identification of a Thyroid Hormone Binding Site in Hsp90 with Implications for Its Interaction with Thyroid Hormone Receptor Beta.

While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb), interacts with Hsp90 to control hormonal perception and signaling. Higher Hsp90 expression in mouse fibroblasts was elicited by the addition of triiodothyronine (T3). T3 bound to Hsp90 and enhanced adenosine triphosphate (ATP) binding of Hsp90 due to a specific binding site for T3, as identified by molecular docking experiments. The binding of TRb to Hsp90 was prevented by T3 or by the thyroid mimetic sobetirome. Purified recombinant TRb trapped Hsp90 from cell lysate or purified Hsp90 in pull-down experiments. The affinity of Hsp90 for TRb was 124 nM. Furthermore, T3 induced the release of bound TRb from Hsp90, which was shown by streptavidin-conjugated quantum dot (SAv-QD) masking assay. The data indicate that the T3 interaction with TRb and Hsp90 may be an amplifier of the cellular stress response by blocking Hsp90 activity.

Triiodothyronine Induces Thyroid Hormone Receptor Beta Release from Hsp90 and Triggers Cellular Stress Response

While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb) interacts with Hsp90 to control hormonal perception and signaling. Treatment with high Triiodothyronine (T3) concentrations induced higher Hsp90 expression in mouse fibroblasts. We can show by microarray-based displacement assay, micro-scale thermophoresis, streptavidin-conjugated quantum dot (SAv-QDs) masking assay or 8-anilino-1-naphthalene sulfone (ANS) fluorescence that T3 enhanced adenosine triphosphate (ATP) binding of Hsp90, while microarray-based interaction analyses revealed that Hsp90 bound directly TRb and was released by T3. The dose-dependent binding of Hsp90 to TRb revealed an affinity of 5 nM. The affinity near the physiological range suggests that the release of TRb induced by T3 additionally influences chaperone activity of Hsp90 when T3 increased over normal concentrations. The observation is that T3 interaction with TRb and Hsp90 is an amplifier of the cellular stress response.

A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. The application of mechanical forces to cultured cells is often performed using specialized systems that lack the flexibility and throughput of other biological techniques. In this study, we developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously. Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the increase in reprograming-related gene expression in mouse fibroblasts when combined with mechanical loading. Taken together, our findings demonstrate a new powerful tool for investigating the mechanobiological mechanisms of disease and performing drug screening in the presence of applied mechanical load.

Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage.

Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, was reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing with an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originated from cells transiently expressing Oct4, whereas ablation of Oct4+ cells prevented iNSC formation. Lastly, an alternative transdifferentiation cocktail that lacks Oct4 and was reportedly unable to support induced pluripotency yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation from fibroblasts using OKSM and other pluripotency-related reprogramming factors requires passage through a transient iPSC state.

Brief Report: Fibrosis Caused by Loss of PTEN Expression in Mouse Fibroblasts Is Crucially Dependent on CCN2

ObjectiveProtein phosphatase and tensin homolog (PTEN) expression is reduced in dermal fibroblasts isolated from patients with diffuse cutaneous systemic sclerosis, a fibrotic autoimmune disease. In support of this finding, deletion of the PTEN gene in the dermal fibroblasts of mice has been shown to result in skin fibrosis and in vivo overexpression of connective tissue growth factor (CTGF; CCN2), a proadhesive matricellular protein; however, whether CCN2 is required for the fibrosis caused by loss of PTEN is unclear. This study was undertaken to investigate the role of CCN2 in fibrosis caused by reduced PTEN expression.MethodsWe generated conditional knockout mice in which PTEN was deleted in fibroblasts, either alone or in combination with CCN2. Skin samples were collected for histologic examination, immunohistochemical analysis, and collagen assay.ResultsLoss of CCN2 resulted in resistance to the increases in collagen production and myofibroblast recruitment that are caused by loss of PTEN. CCN2 deficiency did not impair Akt phosphorylation or the increases in the intensity of proliferating cell nuclear antigen staining that were caused by loss of PTEN.ConclusionThese data are consistent with the notion that CCN2 is required for particular aspects of the fibroproliferative response; therapeutic strategies blocking CCN2 may be of clinical benefit in combating fibrotic disease.

Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.

Sorsby Fundus Dystrophy-Related Mutation in Tissue Inhibitor of Metalloproteinases-3 Impairs Regulation of Its Expression in Mouse Fibroblasts

Mutations in the C-terminal domain of tissue inhibitor of metalloproteinases-3 (TIMP-3) are linked with Sorsby fundus dystrophy (autosomal dominant retinal dystrophy). Experiments on timp-3 gene knock-out and knock-in mice, carriers of dystrophy-linked Timp-3S156C mutation, showed that expression of mutant Timp-3S156C, in contrast to the normal one, is not regulated by phorbol-myristate-12-acetate and is retained at a high level in a medium without FCS. The mutant protein is not transported into the nucleus, while fibroblasts expressing it survive without FCS and other growth factors. It is hypothesized that normal Timp-3 is involved in the regulation of expression of its own timp-3 gene and, probably, other genes associated with growth and proliferation. The absence of this characteristic in mutant Timp-3S156C is presumably responsible for its accumulation in the extracellular matrix.

Met-HGF/SF: tumorigenesis, invasion and metastasis.

Hepatocyte growth factor/scatter factor (HGF/SF) is synthesized by mesenchymal cells and is a paracrine effector of cells, predominantly epithelial, that express the Met tyrosine kinase receptor. We have demonstrated that autocrine Met-HGF/SF expression in mouse fibroblasts results in transformation and tumorigenesis. HGF/SF-treated cells expressing Met can respond in a variety of ways: mitogenically, by scattering (motility), and by forming branching tubules in gel matrices (branching morphogenesis). HGF/SF also induces in vitro invasiveness and is angiogenic in in vivo assays. A human cell line and several mouse cell lines that we have constructed to express Met-HGF/SF in an autocrine fashion are tumorigenic, invasive and metastatic in athymic nude mice. Thus, the very complex process of invasion and metastasis can be mediated by a ligand-receptor signalling pathway, and the cell lines we have developed provide important model systems for identifying the signalling molecules that mediate these phenotypes: For example Met-HGF/SF signalling activates the urokinase plasminogen proteolysis network, thus coupling this signal transduction pathway to the proteases that mediate dissolution of the extracellular matrix. Branching morphogenesis, mediated by Met-HGF/SF signalling, is dependent on this process, as well as the formation of cell-cell junctions and interaction with the extracellular matrix. We have proposed a hypothesis for the role of Met and downstream signalling molecules in generating normal ducts and lumenal structures, as well as a model for how interruption of this signalling leads to abnormal malignant progression. Is Met involved in human cancer? Human sarcomas often inappropriately express Met, suggesting that it is an important oncogene in these cancers, and an increasing number of reports have implicated Met-HGF/SF signalling in a variety of human cancers.

RETRACTED ARTICLE: Sorsby fundus dystrophy-related mutation in tissue inhibitor of metalloproteinases-3 impairs regulation of its expression in mouse fibroblasts

Mutations in the C-terminal domain of tissue inhibitor of metalloproteinases-3 (TIMP-3) are linked with Sorsby fundus dystrophy (autosomal dominant retinal dystrophy). Experiments on timp-3 gene knock-out and knock-in mice, carriers of dystrophy-linked Timp-3S156C mutation, showed that expression of mutant Timp-3S156C, in contrast to the normal one, is not regulated by phorbol-myristate-12-acetate and is retained at a high level in a medium without FCS. The mutant protein is not transported into the nucleus, while fibroblasts expressing it survive without FCS and other growth factors. It is hypothesized that normal Timp-3 is involved in the regulation of expression of its own timp-3 gene and, probably, other genes associated with growth and proliferation. The absence of this characteristic in mutant Timp-3S156C is presumably responsible for its accumulation in the extracellular matrix.

Regulation of membrane trafficking by a novel Cdc42-related protein in Caenorhabditis elegans epithelial cells.

Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C(6)-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines.

BackgroundMatrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown.MethodsTo investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential.ResultsCompetitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed.ConclusionWe have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

Polyomavirus tumorantigens have a profound effect on gene expression in mouse fibroblasts.

Polyomavirus (Py) large and small tumorantigens together are competent to induce S phase in growth-arrested mouse fibroblasts. The capacity of the large tumorantigen to bind the pocket proteins, pRB, p130 and p107, is important for the transactivation of DNA synthesis enzymes and the cyclins E and A, while the interference of small tumorantigen with protein phosphatase PP2A causes a destabilization of the cdk2 inhibitor p27, and thus leads to strong cyclin E- and cyclin A-dependent cdk2 activity. Py small tumorantigen, in addition, is able to transactivate cyclin A. Hence, this protein might have a much wider effect on gene expression in arrested mouse fibroblasts than hitherto suspected. This may have a profound part in the known capacity of Py to form tumors in mice. Therefore, it was interesting to gain an insight into the spectrum of transcriptional deregulation by Py tumorantigens. Accordingly, we performed microarray analysis of quiescent mouse fibroblasts in the absence and presence of small or large tumorantigen. We found that the viral proteins can induce or repress a great variety of genes beyond those involved in the S phase induction and DNA synthesis. The results of the microarray analysis were confirmed for selected genes by several methods, including real-time PCR. Interestingly, a mutation of the binding site for pocket proteins in case of LT and for PP2A in case of ST has a variable effect on the deregulation of genes by the viral proteins depending on the gene in question. In fact, some genes are transactivated by LT as well as ST completely independent of an interaction with their major cellular targets, pocket proteins and PP2A, respectively.

Keratinocyte Growth Inhibition by High-Dose Epidermal Growth Factor Is Mediated by Transforming Growth Factor β Autoinduction: A Negative Feedback Mechanism for Keratinocyte Growth

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

Growth-regulated antisense transcription of the mouse thymidine kinase gene.

The expression of the salvage pathway enzyme thymidine kinase (TK) is very low in resting mammalian cells, but increases dramatically when growth-stimulated cells enter S phase. The 30-fold rise in TK mRNA levels in response to growth factors is due to a well-characterized transcriptional activation and less defined post-transcriptional mechanisms. A minigene containing the murine TK promoter and the TK cDNA showed a 3-fold increase in TK mRNA levels after growth induction in stably transfected mouse TK-deficient L fibroblasts. Introduction of the first three TK introns resulted in a 10-fold regulation of TK expression which was predominantly due to repressed TK mRNA levels in serum-deprived cells. Removal of intron 3 from this construct or replacement of the TK promoter by a constitutive SV40 promoter led to a reduced, but still significant increase in TK mRNA levels during the onset of proliferation. These results indicate that both the TK promoter and specific TK introns contribute independently to the growth-dependent regulation of TK mRNA expression. To examine the regulatory mechanisms in more detail we analyzed TK transcription rates and steady-state levels of nuclear transcripts from an SV40 promoter-driven minigene that contains introns 2 and 3 of the TK gene. Using a set of single-stranded probes we detected TK-specific antisense transcription that was up-regulated in resting cells. Similarly, antisense transcription of the endogenous TK gene in Swiss 3T3 cells rose during serum deprivation while sense transcription was regulated in the opposite way. Luciferase reporter assays revealed the presence of a putative antisense promoter in intron 3 of the murine TK gene. These results suggest a negative role for intron-dependent antisense transcription in the regulation of TK mRNA expression in mouse fibroblasts.

A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators.

We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases-Ipl1 and aurora, respectively-mutations in which cause disruption of chromosome segregation. We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation.

E5 proteins of human papillomavirus types 11 and 16 transactivate the c-fos promoter through the NF1 binding element.

Human papillomavirus type 11 (HPV-11) and HPV-16 contain an E5 gene that can induce c-fos gene expression in mouse fibroblasts. This study investigated the human c-fos promoter characteristics by mapping the c-fos promoter sequence with several deletion and point mutants that confer responsiveness to E5 of HPV-11 or HPV-16. The mutant studies show that NF1 binding sequences within the c-fos promoter were crucial for the induction of the c-fos gene by E5, and the gel shift assay study suggested that E5 of both HPV-11 and HPV-16 is associated, perhaps indirectly, with this NF1 element in the transactivation of the human c-fos promoter. Using an inducible system, we demonstrate that increased induction of the HPV-11 E5 gene in cells led to increased transactivation of the NF1 element. In addition, the transactivating activity of a series of HPV-11 E5 mutants on the NF1 element had a strong correlation with their respective transforming activities.

Distinct Troponin T Genes Are Expressed in Embryonic/Larval Tail Striated Muscle and Adult Body Wall Smooth Muscle of Ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the sessile adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actomyosin Mg2+-ATPase activity in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only <60% identity with each other. Northern blotting and whole mount in situ hybridization revealed that these isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.

Identification of a Murine TEF-1-related Gene Expressed after Mitogenic Stimulation of Quiescent Fibroblasts and during Myogenic Differentiation

Fibroblast growth factor (FGF)-1 binding to cell surface receptors stimulates an intracellular signaling pathway that ultimately promotes the transcriptional activation of specific genes. We have used a mRNA differential display method to identify FGF-1-inducible genes in mouse NIH 3T3 fibroblasts. Here, we report that one of these genes, FGF-regulated (FR)-19, is predicted to encode a member of the transcriptional enhancer factor (TEF)-1 family of structurally related DNA-binding proteins. Specifically, the deduced FR-19 amino acid sequence has approximately89, 77, and 68% overall identity to chicken TEF-1A, mouse TEF-1, and mouse embryonic TEA domain-containing factor, respectively. Gel mobility shift experiments indicate that FR-19, like TEF-1, can bind the GT-IIC motif found in the SV40 enhancer. The FR-19 gene maps in the distal region of mouse chromosome 6, and analysis of several FR-19 cDNA clones indicates that at least two FR-19 isoforms may be expressed from this locus. FGF-1 induction of FR-19 mRNA expression in mouse fibroblasts is first detectable at 4 h after FGF-1 addition and is dependent on de novo RNA and protein synthesis. FGF-2, calf serum, platelet-derived growth factor-BB, and phorbol 12-myristate 13-acetate can also induce FR-19 mRNA levels. We have also found that FR-19 mRNA expression increases during mouse C2C12 myoblast differentiation in vitro. The FR-19 gene is expressed in vivo in a tissue-specific manner, with a relatively high level detected in lung. These results indicate that increased expression of a TEF-1-related protein may be important for both mitogen-stimulated fibroblast proliferation and skeletal muscle cell differentiation.