The myth of Chinese Barbies: eating disorders in China including Hong Kong.

Much of the literature on eating disorders deals with Western subjects. Although the majority of those seen in clinical settings are Caucasians, reports from Asia suggest that anorexia nervosa and bulimia nervosa do occur in the Chinese, sparking debate as to whether or not it is the result of Westernization. This project begins with a review of the current literature on eating disorders in Chinese populations and the role of culture as a mediating factor. A psychodynamic conceptualization and the potential role of traumatic experiences are explored in the emergence of pathological eating habits. Research suggests that applying Western models for Chinese subjects with eating disorders may not always be appropriate.

The retinoblastoma gene family members pRB and p107 coactivate the AP-1-dependent mouse tissue factor promoter in fibroblasts.

Serum-stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of blood coagulation. This requires the rapid entry of c-Fos into specific AP-1 DNA-binding complexes and can be strongly inhibited by the adenovirus EIA 12S gene product. In this study, we utilized a panel of E1A mutants deficient in cellular protein binding to analyse the molecular basis for EIA inhibition of a minimal, c-Fos-dependent TF promoter/ reporter construct in mouse AKR-2B fibroblasts. Mutations which impaired binding of the retinoblastoma tumor suppressor protein family members pRB, p107, and p130 relieved E1A-mediated inhibition of transcription in response to serum-stimulation or c-Fos overexpression. Inhibition was restricted to the G0 to G1 transition, consistent with the specificity of E1A for hypophosphorylated forms of RB proteins. Although E1A mutants deficient in CBP/p300 binding retained the ability to inhibit TF transcription, deletion of the amino-terminal portion of the CBP/p300 interaction domain was required to permit rescue of TF promoter activity by coexpression of pRB. Moreover, ectopic p107 could effectively substitute for pRB in relieving E1A-mediated repression. In primary mouse embryo fibroblasts, activity of the minimal AP-1-dependent TF promoter was suppressed in Rb(-/-) cells compared to parallel Rb(+/-) and Rb(+/+) transfectants. Ectopic expression of either pRB or p107 markedly enhanced TF promoter activity in Rb(-/-) fibroblasts. Collectively, these data imply that pRB and p107 can cooperate with c-Fos to activate TF gene transcription in fibroblasts and suggest a requirement for another, as yet unidentified, E1A-binding protein.

Altered sensitivity to single-strand-specific reagents associated with the genomic vascular smooth muscle alpha-actin promoter during myofibroblast differentiation.

Stimulation of quiescent AKR-2B mouse fibroblasts with transforming growth factor beta1 results in uniform conversion to a myofibroblast-like phenotype as judged by a rapid accumulation of smooth muscle alpha-actin mRNA and protein. Because transcriptional regulation of the smooth muscle alpha-actin gene in these cells might be mediated by single-stranded DNA-binding proteins, we have examined the sensitivity of genomic DNA to chemical reagents with specificity for unpaired bases in a region of the promoter previously implicated in Puralpha, Purbeta, and MSY1 binding in vitro (Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). Our data reveal specific differences between purified DNA treated in vitro and nucleoprotein complexes treated in living cells. Although some differences were observed in quiescent cells, treatment with transforming growth factor beta1 resulted in the development of additional sensitivity within 1 h. This enhancement was most pronounced in bases immediately upstream of an MCAT enhancer element-containing polypurine-polypyrimidine tract. A TATA-proximal element of similar base distribution showed no such hyperreactivities. These results suggest that activation of the endogenous smooth muscle alpha-actin gene during myofibroblast conversion is accompanied by specific structural changes in the promoter that are consistent with a decline in single-stranded DNA repressor protein binding.

The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1 specifically interact with an exon 3-derived mouse vascular smooth muscle alpha-actin messenger RNA sequence.

Amino acids 44-53 of mouse vascular smooth muscle alpha-actin are encoded by a region of exon 3 that bears structural similarity to an essential MCAT enhancer element in the 5' promoter of the gene. The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, interact with each other and with opposite strands of the enhancer to repress transcription in fibroblasts (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436; Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). In this study, we employed both recombinant and fibroblast-derived proteins to demonstrate that all three proteins specifically interact with the mRNA counterpart of the exon 3 sequence in cell-free binding assays. When placed in the 5'-untranslated region of a reporter mRNA, the exon 3-derived sequence suppressed mRNA translation in transfected fibroblasts. Translational efficiency was restored by mutations that impaired mRNA binding of Puralpha, Purbeta, and MSY1, implying that these proteins can also participate in messenger ribonucleoprotein formation in living cells. Additionally, primary structure determinants required for interaction of Purbeta with single-stranded DNA, mRNA, and protein ligands were mapped by deletion mutagenesis. These experiments reveal highly specific protein-mRNA interactions that are potentially important in regulating expression of the vascular smooth muscle alpha-actin gene in fibroblasts.

Molecular interactions between single-stranded DNA-binding proteins associated with an essential MCAT element in the mouse smooth muscle alpha-actin promoter.

Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts.

Sequence of cDNAs encoding components of vascular actin single-stranded DNA-binding factor 2 establish identity to Puralpha and Purbeta.

Transcriptional repression of the mouse vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts is mediated, in part, by the interaction of two single-stranded DNA binding activities with opposite strands of an essential transcription enhancer factor-1 recognition element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436). One of these activities, previously designated vascular actin single-stranded DNA-binding factor 2 includes two distinct polypeptides (p44 and p46) which specifically interact with the purine-rich strand of both the enhancer and a related element in a protein coding exon of the gene (Kelm, R. J., Jr., Sun, S., Strauch, A. R., and Getz, M. J. (1996) J. Biol. Chem. 271, 24278-24285). Expression screening of a mouse lung cDNA library with a vascular actin single-stranded DNA-binding factor 2 recognition element has now resulted in the isolation of two distinct cDNA clones that encode p46 and p44. One of these proteins is identical to Puralpha, a retinoblastoma-binding protein previously implicated in both transcriptional activation and DNA replication. The other is a related family member, presumably Purbeta. Comparative band shift and Southwestern blot analyses conducted with cellular p46, p44, and cloned Pur proteins synthesized in vitro and in vivo, establish identity of p46 with Puralpha and p44 with Purbeta. This study implicates Puralpha and/or Purbeta in the control of vascular smooth muscle alpha-actin gene transcription.

A c-Fos- and E1A-interacting component of the tissue factor basal promoter complex mediates synergistic activation of transcription by transforming growth factor-beta1.

Stimulation of quiescent mouse fibroblasts with TGF-beta1 and certain other growth factors result in cooperative activation of tissue factor (TF) gene transcription, an event accompanied by the rapid entry of c-Fos into specific AP-1 DNA-binding complexes (Felts et al. (1995) Biochemistry 34, 12355-12362). Here, we demonstrate that the ability of TGF-beta1 to synergistically activate TF transcription in serum-stimulated fibroblasts is dependent upon both c-Fos and a promoter-specific factor with functional properties characteristic of transcriptional coactivators. Inhibition of TF promoter activity by an adenovirus E1A mutant deleted in an essential CREB binding protein (CBP) interaction domain suggests that this factor is distinct from the CBP/p300 family of transcriptional coactivators. Importantly, the ability of this factor to mediate molecular interactions with c-Fos required for transcriptional synergism is directly linked to TGF-beta1 signaling. These data suggest a model in which a component of the TF basal transcription complex functions to integrate multiple signaling pathways required for full transcriptional activation of TF in fibroblasts.

Expression of tissue factor in tumor stroma correlates with progression to invasive human breast cancer: paracrine regulation by carcinoma cell-derived members of the transforming growth factor beta family.

Tissue factor (TF), the cellular initiator of the protease blood coagulation cascade, has been shown to be expressed in a variety of solid tumors, particularly those of epithelial origin. However, the mechanisms that mediate TF expression in tumors, as well as the clinical implications of this expression, remain largely unknown. In this study, we examined the cytological distribution of TF in normal human breast tissue and breast carcinomas. Epithelial cells exhibited TF immunoreactivity with little obvious correlation with malignant progression from in situ lesions to invasive cancer. However, there was a strong correlation between progression to invasive cancer and the expression of TF antigen in cellular components of the stroma. TF-positive cells were particularly abundant in close proximity to infiltrating tumor cells and included both macrophages and myofibroblasts, as determined by double-immunofluorescent staining for TF and cell type-specific marker proteins. Double-immunofluorescent staining for TF and transforming growth factor beta (TGF-beta) revealed TGF-beta immunoreactivity both in tumor cells and in the extracellular matrix surrounding TF-positive stromal cells. To test the role of carcinoma cell-derived growth factors in the regulation of stromal cell TF activity, we examined the ability of conditioned media (CM) from breast carcinoma cell lines to stimulate TF activity in myofibroblast-like cells in vitro. Extracts from myofibroblasts exposed to CM displayed strong TF procoagulant activity. However, extracts from cells exposed to unconditioned media or CM pretreated with anti-TGF-beta antibodies did not. The induction of TF activity was also observed upon treatment of indicator cells with recombinant TGF-beta isoforms. Collectively, these data indicate that the recruitment and/or activation of TF-expressing stromal cells is an early event in progression to invasive breast cancer and likely occurs, in part, as a paracrine response to tumor cell-derived members of the TGF-beta family of growth factors.

Repression of transcriptional enhancer factor-1 and activator protein-1-dependent enhancer activity by vascular actin single-stranded DNA binding factor 2.

Transcriptional repression of the murine vascular smooth muscle alpha-actin gene in fibroblasts results from the interaction of two sequence-specific single-stranded DNA binding activities (VACssBF1 and VACssBF2) with opposite strands of an essential transcriptional enhancer factor-1 (TEF-1) element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436). Here, we identify a sequence element located within a protein-coding exon of the gene that bears structural similarity with the TEF-1 enhancer. This includes a 30-base pair region of purine-pyrimidine asymmetry encompassing a perfect 6-base pair GGAATG TEF-1 recognition motif. Unlike the enhancer, however, the exon sequence exhibits no TEF-1 binding activity nor does the pyrimidine-rich strand bind VACssBF1. However, VACssBF2 interacts equally well with the purine-rich strand of both the enhancer and the exon sequence. To test the ability of VACssBF2 to independently repress transcription, the exon sequence was placed upstream of a deletionally activated promoter containing an intact TEF-1 binding site. The exon sequence repressed promoter activity, whereas a mutant deficient in VACssBF2 binding did not. Moreover, VACssBF2 similarly repressed activator protein-1-dependent transcription of a heterologous tissue factor promoter. These results suggest that VACssBF2 possesses an intrinsic ability to disrupt enhancer function independently of the enhancer-binding proteins involved.

Tissue factor gene transcription in serum-stimulated fibroblasts is mediated by recruitment of c-Fos into specific AP-1 DNA-binding complexes.

Serum stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of the protease cascade leading to blood coagulation. In this study, we demonstrate that two AP-1 DNA-binding elements located 200-220 bp upstream of the transcription start site are both necessary and sufficient to confer serum inducibility to the TF gene promoter in fibroblasts. Analysis of AP-1 DNA-binding complexes indicates that the predominant form of AP-1 activity in quiescent cells consists of an unidentified Fos-related protein and JunD. While c-Fos is notably absent from these preexisting complexes, serum stimulation results in the rapid entry of c-Fos into the TF AP-1 DNA-binding complexes. A similar induction of c-Fos DNA-binding activity occurs in cells treated with recombinant growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). Importantly, overexpression of JunD and c-Fos abrogates the requirement for serum in the stimulation of TF promoter activity in fibroblasts. Together, these data indicate that the entry of c-Fos into heterodimeric AP-1 DNA-binding complexes with JunD is a key event underlying serum-stimulated transcription of the TF gene in fibroblasts.

Plasticity of vascular smooth muscle alpha-actin gene transcription. Characterization of multiple, single-, and double-strand specific DNA-binding proteins in myoblasts and fibroblasts.

Transcriptional activity of the mouse vascular smooth muscle (VSM) alpha-actin promoter was governed by both cell type and developmental stage-specific mechanisms. A purine-rich motif (PrM) located as -181 to -176 in the promoter was absolutely required for activation in mouse AKR-2B embryonic fibroblasts and partially contributed to activation in undifferentiated mouse BC3H1 myoblasts. Transcriptional enhancer factor 1 recognized the PrM and cooperated with other promoter-binding proteins to regulate serum growth factor-dependent transcription in both myoblasts and fibroblasts. Two distinct protein factors (VAC-ssBF1 and VAC-ssBF2) also were identified that bound sequence-specifically to single-stranded oligonucleotide probes that spanned both the PrM and a closely positioned negative regulatory element. VAC-ssBF1 and BF2 binding activity was detected in undifferentiated myoblasts, embryonic fibroblasts, and several smooth muscle tissues in the mouse and human. A myoblast-specific protein (VAC-RF1) also was detected that bound double-stranded probes containing a CArG-like sequence that previously was shown to impart strong, cell type specific repression. The binding activity of transcription enhancer factor 1, VAC-RF1, and VAC-ssBF1 was significantly diminished when confluent BC3H1 myoblasts differentiated into myocytes and expressed VSM alpha-actin mRNA after exposure to serum-free medium. The results indicated that cell type-specific control of the VSM alpha-actin gene promoter required the participation of multiple DNA-binding proteins, including two that were enriched in smooth muscle and had preferential affinity for single-stranded DNA.

Negative regulation of the vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts: disruption of enhancer function by sequence-specific single-stranded-DNA-binding proteins.

Transcriptional activation and repression of the vascular smooth muscle (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximately 30-bp polypurine-polypyrimidine tract. This region contains binding sites for an essential transcription-activating protein, identified as transcriptional enhancer factor I (TEF-1), and two tissue-restrictive, sequence-specific, single-stranded-DNA-binding activities termed VACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-binding activity, while VACssBF1 and VACssBF2 have little, if any, affinity for double-stranded DNA. Site-specific mutagenesis experiments demonstrate that the determinants of VACssBF1 and VACssBF2 binding lie on opposite strands of the DNA helix and include the TEF-1 recognition sequence. Functional analysis of this region reveals that the CCAAT box-binding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in activating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. Importantly, replacement of the TEF-1-binding site with that for NF-Y diminishes the ability of VACssBF1 and VACssBF2 to bind to separated single strands. Additional activating mutations have been identified which lie outside of the TEF-1-binding site but which also impair single-stranded-DNA-binding activity. These data support a model in which VACssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcription by stabilizing a local single-stranded-DNA conformation, thus precluding double-stranded-DNA binding by the essential transcriptional activator TEF-1.

Activation of c-fos gene expression by a kinase-deficient epidermal growth factor receptor.

The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR) has been shown to be responsible for many of the pleiotropic intracellular effects resulting from ligand stimulation [W.S. Chen, C.S. Lazar, M. Poenie, R.Y. Tsien, G.N. Gill, and M.G. Rosenfeld, Nature (London) 328:820-823, 1987; A.M. Honegger, D. Szapary, A. Schmidt, R. Lyall, E. Van Obberghen, T.J. Dull, A. Ulrich, and J. Schlessinger, Mol. Cell. Biol. 7:4568-4571, 1987]. Recently, however, it has been shown that addition of ligand to cells expressing kinase-defective EGFR mutants can result in the phosphorylation of mitogen-activated protein kinase (R. Campos-González and J.R. Glenney, Jr., J. Biol. Chem. 267:14535-14538, 1992; E. Selva, D.L. Raden, and R.J. Davis, J. Biol. Chem. 268:2250-2254, 1993), as well as stimulation of DNA synthesis (K.J. Coker, J.V. Staros, and C.A. Guyer, Proc. Natl. Acad. Sci. USA 91:6967-6971, 1994). Moreover, mitogen-activated protein kinase has been shown to phosphorylate the transcription factor p62TCF in vitro, leading to enhanced ternary complex formation between p62TCF, p67SRF, and the c-fos serum response element (SRE) [H. Gille, A.D. Sharrocks, and P.E. Shaw, Nature (London) 358:414-417, 1992]. On the basis of these observations, we have investigated the possibility that the intrinsic tyrosine kinase activity of the EGFR may not be necessary for transcriptional activation mediated via p62TCF. Here, we demonstrate that a kinase-defective EGFR mutant can signal ligand-induced expression of c-fos protein and that a significant component of this induction appears to be mediated at the transcriptional level. Investigation of transcriptional activation mediated by the c-fos SRE shows that this response is impaired by mutations in the SRE which eliminate binding of p62(TCF). These data indicate that information inherent in the structure of the EGFR can be accessed by ligand stimulation independent of the receptor's catalytic kinase function.

Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.

Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways.

Positive and negative cis-acting regulatory elements mediate expression of the mouse vascular smooth muscle alpha-actin gene.

Segments of the 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene were assayed for promoter activity in transfected mouse BC3H1 myogenic cells and AKR-2B embryonic fibroblasts. The region between -150 and -191 that functions as a positive transcriptional element in myogenic and fibroblastic cells contains a mammalian-specific inverted CC(A/T)6GG-type consensus sequence. Expression was restricted to fully differentiated myogenic cells when an additional sequence spanning -191 to -224 was included in reporter gene constructs. This 33-base pair (bp) negative regulatory element is 70% conserved between the mouse and human genes and contains a 10-bp motif at its 3' end that only partially resembles a CC(A/T)6GG element. Retention of a GGGA motif at the 3' boundary of the 33-bp region is sufficient to maintain full transcriptional repression in fibroblasts and is partly responsible for repression in undifferentiated myoblasts. Complete muscle tissue-restrictive expression requires an additional 8 bp from the CC(A/T)6GG-like element immediately 5' to the GGGA motif, since replacement of this region with an unrelated 10-bp sequence completely eliminated restrictive transcriptional behavior in undifferentiated myoblasts. The distal portion of the 5'-flanking region between -224 and -1074 contains six E-box motifs (CANNTG) and mediates high level transcription only in postconfluent BC3H1 myoblasts. Analysis of reporter gene constructs including either the proximal E-box at -240 or all six E-boxes indicate that the five distal E-boxes are not required for high level transcription. A 724-bp segment of the 5'-flanking region consisting of the proximal E-box flanked upstream by a mammalian-specific 352-bp region was sufficient for maximal transcriptional activation in postconfluent BC3H1 myoblasts. Deletion of the 352-bp region restricts the early transcriptional response to high cell density in temporal studies of promoter activity during BC3H1 myogenic cell differentiation.

Cloning of murine tissue factor and regulation of gene expression by transforming growth factor type beta 1.

We have cloned a serum- and cycloheximide-inducible mRNA from AKR-2B murine fibroblasts which encodes a protein with significant sequence similarity to human tissue factor, a cellular initiator of the blood coagulation cascade. Information derived from this clone was used to establish the presence of a virtually identical sequence in mouse brain. Most importantly, cDNA-directed expression in a quail fibroblast cell line produced high levels of tissue factor procoagulant activity, confirming the identity of this protein as murine tissue factor. Additional studies demonstrate that transforming growth factor type beta 1 stimulates tissue factor gene transcription and is a potent inducer of tissue factor procoagulant activity in fibroblasts. Other tested mitogens such as platelet-derived growth factor, epidermal growth factor, and insulin were weak inducers. These results may reflect a role for transforming growth factor beta 1 in the maintenance of hemostasis or, alternatively, a role for tissue factor in cellular functions unrelated to blood coagulation.

Cooperative stimulation of specific gene transcription by epidermal growth factor and transforming growth factor type beta 1.

Transforming growth factor type beta 1 (TGF-beta 1) is a pleiotropic regulator of cell growth and differentiation which can potentiate or otherwise modify cellular responses to different growth factors such as epidermal growth factor (EGF). Since cellular responses to peptide growth factors are mediated, in part, through the regulation of specific gene expression, we have studied the effect of TGF-beta 1 on the EGF-dependent transcriptional of a diverse panel of genes which included the fibronectin gene, the cytoskeletal beta- and gamma-actin genes, and the c-fos protooncogene. The addition of a combination of TGF-beta 1 (10 ng/ml) and EGF (10 ng/ml) to quiescent AKR-2B cells maintained in serum-free medium resulted in a strong synergistic stimulation of transcription which was not evident using either growth factor individually at concentrations of 20-100 ng/ml. This effect was evident within 10 min and did not require the continual presence of TGF-beta 1 in the culture medium. Increased responsiveness of some gene promoters to EGF stimulation was evident for up to 6 h following a brief 10-min exposure to TGF-beta 1. Similar exposure to either platelet-derived growth factor or fibroblast growth factor had little or no effect on EGF-stimulated transcription. These results indicate that the transcriptional response of specific genes to EGF can be stably altered as a consequence of exposure to TGF-beta 1.

Negative regulation of serum-responsive enhancer elements.

Transcription of the c-fos proto-oncogene and the cytoskeletal actin genes is induced within minutes of the addition of serum growth factors in a variety of cell types. Inhibitors of protein synthesis such as cycloheximide have been shown to dramatically potentiate the transcriptional response, an effect termed 'superinduction'. Although the stimulatory effect of serum has been shown to be transmitted through a cis-acting enhancer sequence termed a serum response element (SRE), the sequence element(s) responsible for mediating the effect of cycloheximide has not been identified. We now report that a synthetic copy of the c-fos SRE is sufficient to confer cycloheximide-dependent inducibility upon a heterologous promoter. This does not require the presence of serum, but several mutations in the SRE that impair serum-inducibility also impair cycloheximide-inducubility. These results imply that serum-responsive enhancer elements are negatively regulated by one or more labile proteins and that both positive and negative regulators of enhancer activity require a functional 'CArG box', a sequence domain previously implicated in muscle-specific transcription.

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.