Ravi P. Misra

A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

Nur77 Is Differentially Modified in PC12 Cells upon Membrane Depolarization and Growth Factor Treatment

The rat pheochromocytoma cell line PC12 can be induced by growth factors to undergo proliferation and neuronal differentiation. These cells also have excitable membranes that can be depolarized by neurotransmitters or elevated levels of extracellular KCl. Treatment of PC12 cells with growth factors or membrane-depolarizing agents rapidly activates the expression of specific genes whose products are thought to mediate the subsequent biological responses. One such gene, nur77, is a member of the steroid and thyroid hormone receptor gene superfamily. We have identified the Nur77 protein and shown that it is synthesized rapidly and transiently in PC12 cells following stimulation, has a short half-life of 30 to 40 min, and is located in both the nucleus and the cytoplasm. Nur77 is posttranslationally modified, primarily by phosphorylation on serine residues. Phosphopeptide analysis reveals that Nur77 is modified differently upon membrane depolarization than after treatment with growth factors. We hypothesize that the activity of Nur77 is regulated by both differential gene expression and posttranslational modification and that these modes of regulation contribute to distinct downstream responses specific to membrane depolarization and growth factor treatment.

Role of the serum response factor in regulating contractile apparatus gene expression and sarcomeric integrity in cardiomyocytes.

The serum response factor (SRF) is a transcriptional regulator required for mesodermal development, including heart formation and function. Previous studies have described the role of SRF in controlling expression of structural genes involved in conferring the myogenic phenotype. Recent studies by us and others have demonstrated embryonic lethal cardiovascular phenotypes in SRF-null animals, but have not directly addressed the mechanistic role of SRF in controlling broad regulatory programs in cardiac cells. In this study, we used a loss-of-function approach to delineate the role of SRF in cardiomyocyte gene expression and function. In SRF-null neonatal cardiomyocytes, we observed severe defects in the contractile apparatus, including Z-disc and stress fiber formation, as well as mislocalization and/or attenuation of sarcomeric proteins. Consistent with this, gene array and reverse transcription-PCR analyses showed down-regulation of genes encoding key cardiac transcriptional regulatory factors and proteins required for the maintenance of sarcomeric structure, function, and regulation. Chromatin immunoprecipitation analysis revealed that at least a subset of these proteins are likely regulated directly by SRF. The results presented here indicate that SRF is an essential coordinator of cardiomyocyte function due to its ability to regulate expression of numerous genes (some previously identified and at least 28 targets newly identified in this study) that are involved in multiple and disparate levels of sarcomeric function and assembly.

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.

Phosphocitrate Inhibits a Basic Calcium Phosphate and Calcium Pyrophosphate Dihydrate Crystal-induced Mitogen-activated Protein Kinase Cascade Signal Transduction Pathway

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fosand c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB’s ability to transactivate. Treatment of cells with PC at concentrations of 10−3 to 10−5 m blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10−3 m, a PC analogue,n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1β induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.

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.

Extrachromosomal DNA in eucaryotes

Eucaryotic extrachromosomal DNAs have been organized into four major classes: (1) Organelle DNAs, (2) plasmid DNAs, (3) amplified genes, and (4) intermediates and/or by-products of DNA transpositions and rearrangements. In this review some of the relatively well-characterized members of each class are described; it is suggested that many of them reflect the complexity and plasticity of eucaryotic genomes.

Gene targeting in the mouse: advances in introduction of transgenes into the genome by homologous recombination.

The ability to stably introduce genes into the germline of animals provides a powerful means to address the genetic basis of physiology. Introduction of genes to generate transgenic animals has facilitated the development of complex genetic models of disease, as well as the in vivo study of gene function. However, one drawback of traditional transgenic technologies in which genes are microinjected into early-stage embryos is that there is little control over where and in how many copies genes are introduced into the genome. The development of animal transgenic technologies, which take advantage of homologous recombination mechanisms and the manipulation of embryonic stem (ES) cells, allows investigators to target and alter specific loci. In mouse transgenic systems, a plethora of sophisticated gene-targeting strategies now permit investigators to manipulate the genome in ways that essentially allow one to introduce virtually any desired change into the genome. Fur-thermore, when coupled with systems that allow for conditional gene expression, these gene-targeting strategies allow both temporal and tissue specific control of alterations to the genome. In the present review we briefly discuss some of the more recent gene-targeting strategies that have been developed to address the limitations of traditional animal transgenesis.

Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in response to serum growth signals

Serum Response Factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth, differentiation, and development. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied, however, less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including an ETS domain binding site, an overlapping Sp1/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces the SRF gene by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation by cells by lysophosphatidic acid (LPA) or whole serum, the SRF promoter is upregulated by a bipartite pathway that requires both an Sp1 factor binding site and the CArG motifs for maximal stimulation. The CArG box-dependent component of this pathway is targeted by Rho mediated signals, and the Sp1 binding site dependent component is targeted by Ras mediated signals.

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

BackgroundTransgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites.ResultsHere we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the α-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern.ConclusionOur results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This facilitates the comparative analysis of lethal alleles and thereby advances our ability to analyze gene function in mammals.