Serum Response Factor Expression Research Articles

A Possible Role for the Cnidarian Homologue of Serum Response Factor in Decision Making by Undifferentiated Cells

We have isolated the serum response factor (SRF) homologue from two hydrozoans, the freshwater polyp Hydra vulgaris and the marine colonial Hydractinia echinata; we have termed the Hydra gene HvSRF and the Hydractinia gene HeSRF. The MADS-box of both genes is identical in sequence and more similar to SRFs of other organisms than to non-SRF MADS-box-containing proteins from other organisms. Within the N terminus of the predicted protein, a motif of 14 amino acids is nearly identical between Hydra and Hydractinia. This motif is absent from other known SRF sequences. In the adult Hydra polyp, SRF is predominantly expressed in cells of the interstitial cell (I-cell) lineage. Expression of SRF ceases when I-cells differentiate into nerve cells, nematocytes, or gland cells. In the course of sexual reproduction in Hydractinia, SRF is expressed in female germ cells. During embryogenesis, SRF transcripts are observed in all blastomeres. Later on, SRF expression is turned off in cells forming the ectodermal layer but further on is expressed in cells of the central cell mass, from which the endodermal epithelial cells and the I-cell lineage originate. Expression eventually becomes restricted to the I-cell lineage. We conclude that hydrozoan SRF is expressed in all these cells, which still have the property for differentiation. In adult Hydra, the abundance of SRF transcripts varies during the day. The pacemaker of this diurnal rhythm is the feeding regime. HvSRF expression decreases by 4 h after feeding and returns to the initial level 12 h after feeding. When feeding is stopped, the cycle of SRF expression persists through the first day when the animals are not fed. It has been shown that feeding partly synchronizes the cell cycle of the epithelial cells but not that of the I-cells. We suggest that the epithelial cells affect SRF expression in I-cells and thereby influence the decision of I-cells to enter a differentiation pathway.

Rho kinases play an obligatory role in vertebrate embryonic organogenesis.

Rho-associated kinases (Rho kinases), which are downstream effectors of RhoA GTPase, regulate diverse cellular functions including actin cytoskeletal organization. We have demonstrated that Rho kinases also direct the early stages of chick and mouse embryonic morphogenesis. We observed that Rho kinase transcripts were enriched in cardiac mesoderm, lateral plate mesoderm and the neural plate. Treatment of neurulating embryos with Y27632, a specific inhibitor of Rho kinases, blocked migration and fusion of the bilateral heart primordia, formation of the brain and neural tube, caudalward movement of Hensen's node, and establishment of normal left-right asymmetry. Moreover, Y27632 induced precocious expression of cardiac alpha-actin, an early marker of cardiomyocyte differentiation, coincident with the upregulated expression of serum response factor and GATA4. In addition, specific antisense oligonucleotides significantly diminished Rho kinase mRNA levels and replicated many of the teratologies induced by Y27632. Thus, our study reveals new biological functions for Rho kinases in regulating major morphogenetic events during early chick and mouse development.

Recruitment of serum response factor and hyperacetylation of histones at smooth muscle-specific regulatory regions during differentiation of a novel P19-derived in vitro smooth muscle differentiation system.

Little is known regarding transcriptional regulatory mechanisms that control the sequential and coordinate expression of genes during smooth muscle cell (SMC) differentiation. To facilitate mechanistic studies of SMC differentiation, we established a novel P19-derived clonal cell line (designated A404) harboring a smooth muscle (SM) alpha-actin promoter/intron-driven puromycin resistance gene. Retinoic acid plus puromycin treatment stimulated rapid differentiation of multipotential A404 cells into SMCs that expressed multiple SMC differentiation marker genes, including the definitive SM-lineage marker SM myosin heavy chain. Using this system, we demonstrated that various transcription factors were upregulated coincidentally with the expression of SMC differentiation marker genes. Of interest, the expression of serum response factor (SRF), whose function is critical for SMC-specific transcription, was high in undifferentiated A404 cells, and it did not increase over the course of differentiation. However, chromatin immunoprecipitation analyses showed that SRF did not bind the target sites of endogenous SMC marker genes in chromatin in undifferentiated cells, but it did in differentiated A404 cells, and it was associated with hyperacetylation of histones H3 and H4. The present studies define a novel cell system for studies of transcriptional regulation during the early stages of SMC differentiation, and using this system, we obtained evidence for the involvement of chromatin remodeling and selective recruitment of SRF to CArG elements in the induction of cell-selective marker genes during SMC differentiation.

Muscle Specificity Encoded by Specific Serum Response Factor-binding Sites

Serum response factor (SRF) is a MADS box transcription factor that regulates muscle-specific and growth factor-inducible genes by binding the consensus sequence CC(A/T)6GG, known as a CArG box. Because SRF expression is not restricted solely to muscle, its expression alone cannot account for the muscle specificity of some of its target genes. To understand further the role of SRF in muscle-specific transcription, we created transgenic mice harboring lacZ transgenes linked to tandem copies of different CArG boxes with flanking sequences. CArG boxes from the SM22 and skeletal alpha-actin promoters directed highly restricted expression in developing smooth, cardiac, and skeletal muscle cells during early embryogenesis. In contrast, the CArG box and flanking sequences from the c-fos promoter directed expression throughout the embryo, with no preference for muscle cells. Systematic swapping of the core and flanking sequences of the SM22 and c-fos CArG boxes revealed that cell type specificity was dictated in large part by sequences immediately flanking the CArG box core. Sequences that directed widespread embryonic expression bound SRF more strongly than those that directed muscle-restricted expression. We conclude that sequence variations among CArG boxes influence cell type specificity of expression and account, at least in part, for the ability of SRF to distinguish between growth factor-inducible and muscle-specific genes in vivo.

Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor.

Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac alpha-actin, skeletal alpha-actin, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.

Senescence represses the nuclear localization of the serum response factor and differentiation regulates its nuclear localization with lineage specificity.

The differentiation of cultured 3T3T mesenchymal stem cells into adipocytes represses growth factor responsiveness by limiting the nuclear localization of the serum response factor (SRF) that binds to and activates the promoters of growth control genes that contain the serum response elements (SRE), such as junB and c-fos. The regulation of SRF nuclear localization by adipocyte differentiation is specific, because we show that adipocyte differentiation does not repress the nuclear localization of six other transacting factors. To determine if repression of growth factor responsiveness that occurs during senescence also represses the nuclear localization of SRF, we studied normal human WI-38 fibroblasts at low versus high population doublings. The results show that SRF localizes to the nucleus of proliferative cells whereas in senescent cells SRF can not be detected in the nucleus. This result is apparent in both immunofluorescence assays and in western blot analysis. We next evaluated the cellular distribution of SRF in selected human tissues to determine whether the loss of proliferative potential in vivo could have a different effect on SRF nuclear localization. We found that in cells of the small bowel mucosa, differentiation modulates SRF nuclear localization in an opposite manner. Minimal SRF expression and nuclear localization is evident in undifferentiated cells at the base of crypts whereas increased SRF expression and nuclear localization is evident in differentiated cells at the surface tip of the villus. These results together establish that regulation of SRF expression and nuclear localization is important in senescence and differentiation in a lineage specific manner.

Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcription-promoting activity.

Increased level and duration of expression in muscle by co-expression of a transactivator using plasmid systems.

Skeletal muscle is an attractive target for gene therapies to treat either local or systemic disorders, as well as for genetic vaccination. An ideal expression system for skeletal muscle would be characterized by high level, extended duration of expression and muscle specificity. Viral promoters, such as the cytomegalovirus (CMV) promoter, produce high levels of transgene expression, which last for only a few days at high levels. Moreover, many promoters lack muscle tissue specificity. A muscle-specific skeletal alpha-actin promoter (SkA) has shown tissue specificity but lower peak activity than that of the CMV promoter in vivo. It has been reported in vitro that serum response factor (SRF) can stimulate the transcriptional activity of some muscle-specific promoters. In this study, we show that co- expression of SRF in vivo is able to up-regulate SkA promoter-driven expression about 10-fold and CMV/SkA chimeric promoter activity by five-fold in both mouse gastrocnemius and tibialis muscle. In addition, co-expression of transactivator with the CMV/SkA chimeric promoter in muscle has produced significantly enhanced duration of expression compared with that shown by the CMV promoter-driven expression system. A dominant negative mutant of SRF, SRFpm, abrogated the enhancement to SkA promoter activity, confirming the specificity of the response. Since all the known muscle-specific promoters contain SRF binding sites, this strategy for enhanced expression may apply to other muscle-specific promoters in vivo.

Persistent Increased DNA-Binding and Expression of Serum Response Factor Occur with Epilepsy-Associated Long-Term Plasticity Changes

We have previously shown that NMDA receptor activation during status epilepticus (SE) is required to produce epilepsy in in vitro and in vivo models. As in human symptomatic epilepsy, the epilepsy in these models is permanent, suggesting that the pathological activation of NMDA receptors causes permanent plasticity changes in the brain. Ca(2+) influx through NMDA receptors is known to transiently activate a key transcription factor, serum response factor (SRF). Thus, we investigated whether this factor, in terms of its expression and ability to bind to the consensus serum response element, was altered long term in the pilocarpine model of epilepsy. In hippocampal nuclear extracts, SRF binding to DNA was significantly increased over saline-injected control rats at 24 hr and at 8 weeks after the onset of SE. This increase was shown to be the result of significantly elevated levels of SRF. DNA binding was also persistently increased in the cortical, but not in the cerebellar, extracts. Hippocampal expression of SRF was localized to neurons using immunohistochemistry. NMDA receptor activation during SE was required for these changes to take place, and the spontaneous seizures seen in epileptic rats did not appear to be responsible for the increase in SRF. The results demonstrate that SRF is persistently elevated after SE in the pilocarpine model of epilepsy and support the theory that long-term gene changes in this model occur and are associated with the long-lasting plasticity changes that are initiated during epileptogenesis.

A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells

Coronary artery smooth muscle (SM) cells originate from proepicardial cells that migrate over the surface of the heart, undergo epithelial to mesenchymal transformation and invade the subepicardial and cardiac matrix. Prior to contact with the heart, proepicardial cells exhibit no expression of smooth muscle markers including SMalphaactin, SM22alpha, calponin, SMgammaactin or SM-myosin heavy chain detectable by RT-PCR or by immunostaining. To identify factors required for coronary smooth muscle differentiation, we excised proepicardial cells from Hamburger-Hamilton stage-17 quail embryos and examined them ex vivo. Proepicardial cells initially formed an epithelial colony that was uniformly positive for cytokeratin, an epicardial marker. Transcripts for flk-1, Nkx 2.5, GATA4 or smooth muscle markers were undetectable, indicating an absence of endothelial, myocardial or preformed smooth muscle cells. By 24 hours, cytokeratin-positive cells became SMalphaactin-positive. Moreover, serum response factor, undetectable in freshly isolated proepicardial cells, became strongly expressed in virtually all epicardial cells. By 72 hours, a subset of epicardial cells exhibited a rearrangement of cytoskeletal actin, focal adhesion formation and acquisition of a motile phenotype. Coordinately with mesenchymal transformation, calponin, SM22alpha and SMgammaactin became expressed. By 5-10 days, SM-myosin heavy chain mRNA was found, by which time nearly all cells had become mesenchymal. RT-PCR showed that large increases in serum response factor expression coincide with smooth muscle differentiation in vitro. Two different dominant-negative serum response factor constructs prevented the appearance of calponin-, SM22alpha- and SMgammaactin-positive cells. By contrast, dominant-negative serum response factor did not block mesenchymal transformation nor significantly reduce the number of cytokeratin-positive cells. These results indicate that the stepwise differentiation of coronary smooth muscle cells from proepicardial cells requires transcriptionally active serum response factor.

The Developmentally Regulated Expression of Serum Response Factor Plays a Key Role in the Control of Smooth Muscle-Specific Genes

Serum response factor (SRF) is a MADS box transcription factor that has been shown to be important in the regulation of a variety of muscle-specific genes. We have previously shown SRF to be a major component of multiplecis/transinteractions found along the smooth muscle γ-actin (SMGA) promoter. In the studies reported here, we have further characterized the role of SRF in the regulation of the SMGA gene in the developing gizzard. EMSA analyses, using nuclear extracts derived from gizzards at various stages in development, showed that the SRF-containing complexes were not present early in gizzard smooth muscle development, but appeared as development progressed. We observed an increase in SRF protein and mRNA levels during gizzard development by Western and Northern blot analyses, with a large increase just preceding an increase in SMGA expression. Thus, changes in SRF DNA-binding activity were paralleled with increased SRF gene expression. Immunohistochemical analyses demonstrated a correspondence of SRF and SMGA expression in differentiating visceral smooth muscle cells (SMCs) during gizzard tissue development. This correspondence of SRF and SMGA expression was also observed in cultured smooth muscle mesenchyme induced to express differentiated gene productsin vitro.In gene transfer experiments with SMGA promoter–luciferase reporter gene constructs we observed four- to fivefold stronger SMGA promoter activity in differentiated SMCs relative to replicating visceral smooth muscle cells. Further, we demonstrate the ability of a dominant negative SRF mutant protein to specifically inhibit transcription of the SMGA promoter in visceral smooth muscle, directly linking SRF with the control of SMGA gene expression. Taken together, these data suggest that SRF plays a prominent role in the developmental regulation of the SMGA gene.

High conservation of the serum response factor within Metazoa: cDNA from the sponge Geodia cydonium

Abstract A cDNA clone encoding the sequence-specific DNA binding protein, serum response factor (SRF), has been isolated and analysed from the marine sponge Geodia cydonium . The 1443 bp long cDNA comprises an open reading frame of 1149 bp, from which an aa sequence with a M r 42,149 can be deduced. Sequence comparisons revealed that the aa sequence shares high homology to human and Xenopus laevis SRFs. Like these vertebrate sequences also the sponge sequence displays the MADS-box motif present in the DNA-binding domain. The SRF, and also other transcription factors, such as ternary complex factors, recognizes the serum response element (CArG box) in promoter regions of a series of cellular immediate-early genes, whose expression is controlled by growth factors. A related CArG box sequence is present in sponge gene, encoding a receptor tyrosine kinase. Expression of SRF is strongly regulated by temperature stress; after incubation of the sponge at high temperature, the 1.6 kb transcript of SRF is downregulated. It is suggested that the SRF is involved in the transcriptional regulatory circuits, which control the response of the animal to altered environmental conditions.

High conservation of the serum response factor within Metazoa: cDNA from the spongeGeodia cydonium

A cDNA clone encoding the sequence-specific DNA binding protein, serum response factor (SRF), has been isolated and analysed from the marine sponge Geodia cydonium . The 1443 bp long cDNA comprises an open reading frame of 1149 bp, from which an aa sequence with a M r 42,149 can be deduced. Sequence comparisons revealed that the aa sequence shares high homology to human and Xenopus laevis SRFs. Like these vertebrate sequences also the sponge sequence displays the MADS-box motif present in the DNA-binding domain. The SRF, and also other transcription factors, such as ternary complex factors, recognizes the serum response element (CArG box) in promoter regions of a series of cellular immediate-early genes, whose expression is controlled by growth factors. A related CArG box sequence is present in sponge gene, encoding a receptor tyrosine kinase. Expression of SRF is strongly regulated by temperature stress; after incubation of the sponge at high temperature, the 1.6 kb transcript of SRF is downregulated. It is suggested that the SRF is involved in the transcriptional regulatory circuits, which control the response of the animal to altered environmental conditions.

Avian Serum Response Factor Expression Restricted Primarily to Muscle Cell Lineages Is Required for α-Actin Gene Transcription

Serum response factor (SRF) gene expression in avian embryonic muscle lineages plays a central role in activating α-actin gene activity. In early stage HH 6 avian embryos, SRF mRNA expression showed strong localization to the neural groove, primitive streak, lateral plate mesoderm, and Hensen's node, while distinct SRF expression was seen later in the neural folds and the somites by HH stage 8. SRF transcripts appeared in the precardiac splanchnic mesoderm in stage HH 9 embryos and was detected at higher levels in the myocardium, somites, and lateral mesoderm of HH 11 embryos. SRF antibody staining demonstrated significant SRF protein accumulation in the myocardium of the developing heart and the myotomal portion of somites. During primary myogenesis in culture, SRF transcripts and nuclear SRF protein content increased about 40-fold, as primary myoblasts withdrew from the cell cycle, reaching their highest levels prior to the upregulation of the skeletal α-actin gene. A dominant-negative SRF mutant, SRFpm1, which inhibited DNA binding, but not dimerization of monomeric SRF subunits, blocked transcriptional activation of a skeletal α-actin promoter–luciferase reporter gene during myogenesis. Transcriptional blockade was reversed by co-transfections of a wild-type SRF expression vector, but was not rescued by the expression of other myogenic factors, such as MyoD and Mef-2C. Thus, SRF displayed an embryonic expression pattern restricted primarily to striated muscle cell lineages, in which increased mass of nuclear SRF was obligatory for α-actin gene transcription.

Expression of the Serum Response Factor Gene Is Regulated by Serum Response Factor Binding Sites

The serum response factor (SRF) is a ubiquitous transcription factor that plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. Notably, SRF has been found to be a key regulator of members of a class of cellular response genes termed immediate-early genes (IEGs), many of which are believed to be involved in regulating cell growth and differentiation. The mechanism by which SRF activates transcription of IEGs in response to mitogenic agents has been extensively studied. Significantly less is known about how expression of the SRF gene itself is mediated. We and others have previously shown that the SRF gene is itself transiently induced by a variety of mitogenic agents and belongs to a class of "delayed" early response genes. We have cloned the SRF promoter and in the present study have analyzed the upstream regulatory sequences involved in mediating serum responsiveness of the SRF gene. Our analysis indicates that inducible SRF expression requires both SRF binding sites located within the first 63 nucleotides upstream from the start site of transcriptional initiation and an Sp1 site located 83 nucleotides upstream from the start site. Maximal transcriptional activity of the promoter also requires two CCAATT box sites located 90 and 123 nucleotides upstream of the start site.

Expression and activity of serum response factor is required for expression of the muscle-determining factor MyoD in both dividing and differentiating mouse C2C12 myoblasts.

To understand the mechanism by which the serum response factor (SRF) is involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the expression of the muscle-determining factor MyoD. Inhibition of SRF activity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the endogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF containing the DNA-binding domain of SRF, which acts as a dominant negative mutant in vivo), or purified anti-SRF antibodies rapidly prevents the expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in proliferating myoblasts but also in myoblasts cultured under differentiating conditions. Additionally, by using a cellular system expressing a glucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have shown that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofluorescence and Northern blot analysis. Taken together these data demonstrate that SRF expression and activity are required for the expression of the muscle-determining factor MyoD.

Activation of Smooth Muscle α-Actin Promoter in ras-Transformed Cells by Treatments with Antimitotic Agents: Correlation with Stimulation of SRF: SRE Mediated Gene Transcription

Smooth muscle alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cells. We have recently shown that serum response factor (SRF) which can bind to the serum response elements (SREs) present in the alpha-actin promoter, can activate alpha-actin promoter activity in ras-transformed cells and suppress transformation by ras. Agents that stimulate SRF expression and alpha-actin promoter activity in ras-transformed cells are expected to be potential candidates as antitumor agents. In this study, we show that treatment of ras-transformed cells with antitumor agents such as taxol, vincristine, vinblastine, colchicine, and nocodazole leads to 5- to 7-fold activation of alpha-actin promoter driven CAT activity, whereas there was very little effect on thymidine kinase promoter driven CAT activity. This activation occurred at subcytotoxic concentrations of these agents and correlated with inhibition of cell cycle progression. Furthermore, these agents stimulated SRF expression in ras-transformed cells, as measured by its SRE binding activity. The increase in alpha-actin expression is accompanied by the restoration of actin filaments into organized bundles. These results suggest a novel mechanism by which antimitotic agents suppress the ras-transformed phenotype.

Serum response factor enhances liver metastasis of colorectal carcinoma via alteration of the E-cadherin/β-catenin complex

Serum response factor (SRF) is a transcription factor that controls cell growth, differentiation, and tumor progression as well as muscle development and function. Reduced expression of cell adhesion molecules has been reported to be associated with tumor metastasis. The aim of this study was to evaluate the expression and a role of SRF in liver metastasis of primary colorectal carcinomas. We examined the expression of SRF, E-cadherin, and beta-catenin by the use of immunochemical staining in 43 cases as a set of primary colorectal carcinomas and liver metastases. We also examined the role of SRF in colorectal carcinoma by overexpression of SRF in a colon cancer cell line. In metastatic carcinoma surgical samples, there was a marked increased expression of SRF as compared to expression in primary colorectal carcinoma surgical samples (P<0.05). E-cadherin expression was significantly decreased in metastatic liver carcinoma samples as compared to primary colorectal carcinoma samples (P<0.001). Frequent nuclear translocation of beta-catenin protein in primary and metastatic carcinoma cells was observed. Overexpression of SRF in SW480 cells resulted in a decreased expression of E-cadherin and an increased expression of non-phosphorylated nuclear beta-catenin. Overexpression of SRF in colorectal carcinoma cells enhanced cell motility and invasiveness. These results indicate that overexpression of SRF in colorectal carcinoma cells is associated with modulation of E-cadherin/beta-catenin expression and may play an important role in colorectal cancer metastasis.

The serum response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated fibroblasts.

Growth factor regulation of c-fos proto-oncogene transcription is mediated by a 20-bp region of dyad symmetry, termed the serum response element. The inner core of this element binds a 67-kDa phosphoprotein, the serum response factor (SRF), that is thought to play a pivotal role in the c-fos transcriptional response. To investigate the mechanism by which SRF regulates c-fos expression, we generated polyclonal anti-SRF antibodies and used these antibodies to analyze the biochemical properties of SRF. These studies indicate that the synthesis of SRF is transient, occurring within 30 min to 4 h after serum stimulation of quiescent fibroblasts. Newly synthesized SRF is transported to the nucleus, where it is increasingly modified by phosphorylation during progression through the cell cycle. Within 2 h of serum stimulation, differentially modified forms of SRF can be distinguished on the basis of the ability to bind a synthetic serum response element. SRF protein exhibits a half-life of greater than 12 h and is predominantly nuclear, with no change occurring in its localization upon serum stimulation. We find that the induction of SRF synthesis is regulated at the transcriptional level and that cytoplasmic SRF mRNA is transiently expressed with somewhat delayed kinetics compared with c-fos mRNA expression. These features of SRF expression suggest a model whereby newly synthesized SRF functions in the shutoff of c-fos transcription.

The Serum Response Factor Is Extensively Modified by Phosphorylation Following Its Synthesis in Serum-Stimulated Fibroblasts

Growth factor regulation of c-fos proto-oncogene transcription is mediated by a 20-bp region of dyad symmetry, termed the serum response element. The inner core of this element binds a 67-kDa phosphoprotein, the serum response factor (SRF), that is thought to play a pivotal role in the c-fos transcriptional response. To investigate the mechanism by which SRF regulates c-fos expression, we generated polyclonal anti-SRF antibodies and used these antibodies to analyze the biochemical properties of SRF. These studies indicate that the synthesis of SRF is transient, occurring within 30 min to 4 h after serum stimulation of quiescent fibroblasts. Newly synthesized SRF is transported to the nucleus, where it is increasingly modified by phosphorylation during progression through the cell cycle. Within 2 h of serum stimulation, differentially modified forms of SRF can be distinguished on the basis of the ability to bind a synthetic serum response element. SRF protein exhibits a half-life of greater than 12 h and is predominantly nuclear, with no change occurring in its localization upon serum stimulation. We find that the induction of SRF synthesis is regulated at the transcriptional level and that cytoplasmic SRF mRNA is transiently expressed with somewhat delayed kinetics compared with c-fos mRNA expression. These features of SRF expression suggest a model whereby newly synthesized SRF functions in the shutoff of c-fos transcription.