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Eupatorium lindleyanum DC. has been used as a functional food in China for a long time. However, the antifibrotic activity of total sesquiterpenoids from Eupatorium lindleyanum DC. (TS-EL) is still unknown. In this study, we discovered that TS-EL reduced the increase in α-smooth muscle actin (α-SMA), type I collagen and fibronectin content, the formation of cell filaments and collagen gel contraction in transforming growth factor-β1-stimulated human lung fibroblasts. Intriguingly, TS-EL did not change the phosphorylation of Smad2/3 and Erk1/2. TS-EL decreased the levels of serum response factor (SRF), a critical transcription factor of α-SMA, and SRF knockdown alleviated the transition of lung myofibroblasts. Furthermore, TS-EL significantly attenuated bleomycin (BLM)-induced lung pathology and collagen deposition and reduced the levels of two profibrotic markers, total lung hydroxyproline and α-SMA. TS-EL also decreased the levels of SRF protein expression in BLM-induced mice. These results suggested that TS-EL attenuates pulmonary fibrosis by inhibiting myofibroblast transition via the downregulation of SRF., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Serum response factor (SRF) cooperates with various co-factors to manage the specification of diverse cell lineages during heart development. Many microRNAs mediate the function of SRF in this process. However, how are miR210 and miR30c involved in the decision of cardiac cell fates remains to be explored. In this study, we found that SRF directly controlled the cardiac expression of miR210. Both miR210 and miR30c blocked the formation of beating cardiomyocyte during embryoid body (EB) differentiation, a cellular model widely used for studying cardiogenesis. Both of anticipated microRNA targets and differentially expressed genes in day8 EBs were systematically determined and enriched with gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and Reactome. Functional enrichments of prediction microRNA targets and down-regulated genes in day8 EBs of miR210 suggested the importance of PI3K-Akt signal and ETS2 in miR210 inhibition of cardiomyocyte differentiation. Similar analyses revealed that miR30c repressed both developmental progress and the adrenergic signaling in cardiomyocytes during the differentiation of EBs. Taken together, SRF directs the expression of miR210 and miR30c, and they repress cardiac development via inhibiting the differentiation of cardiac muscle cell lineage as well as the cell proliferation. Through the regulation of specific microRNAs, the complication of SRF's function in heart development is emphasized., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We recently identified a novel, heterozygous, and non-synonymous ACTC1 mutation (p.Gly247Asp or G247D) in a large, multi-generational family, causing atrial-septal defect followed by late-onset dilated cardiomyopathy (DCM). Molecular dynamics studies revealed possible actin polymerization defects as G247D mutation resides at the juncture of side-chain interaction, which was indeed confirmed by in vitro actin polymerization assays. Since polymerization/de-polymerization is important for the activation of Rho-GTPase-mediated serum response factor (SRF)-signaling, we studied the effect of G247D mutation using luciferase assay. Overexpression of native human ACTC1 in neonatal rat cardiomyocytes (NRVCMs) strongly activated SRF-signaling both in C2C12 cells and NRVCMs, whereas, G247D mutation abolished this activation. Mechanistically, we found reduced GTP-bound Rho-GTPase and increased nuclear localization of globular actin in NRVCMs overexpressing mutant ACTC1 possibly causing inhibition of SRF-signaling activation. In conclusion, our data suggests that human G247D ACTC1 mutation negatively regulates SRF-signaling likely contributing to the late-onset DCM observed in mutation carrier patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The ubiquitin-conjugating enzymes 2C (UBE2C) is an integral component of the ubiquitin proteasome system. UBE2C consists of a conserved core domain containing the catalytic Cys residue and an N-terminal extension. The core domain is required for ubiquitin adduct formation by interacting with the ubiquitin-fold domain in the E1 enzyme, and contributes to the E3 enzyme binding. UBE2C N-terminal extension regulates E3 enzyme activity as a part of an intrinsic inhibitory mechanism. UBE2C is required for the destruction of mitotic cyclins and securin, which are essential for spindle assembly checkpoint and mitotic exit. The UBE2C mRNA and/or protein levels are aberrantly increased in many cancer types with poor clinical outcomes. Accumulation of UBE2C stimulates cell proliferation and anchorage-independent growth. UBE2C transgenic mice are prone to develop spontaneous tumors and carcinogen-induced tumor with evidence of chromosome aneuploidy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Adult stem cells and progenitors are of great interest for their clinical application as well as their potential to reveal deep sensitivities to microenvironmental factors. The bone marrow is a niche for at least two types of stem cells, and the prototype is the hematopoietic stem cell/progenitors (HSC/Ps), which have saved many thousands of patients for several decades now. In bone marrow, HSC/Ps interact functionally with marrow stromal cells that are often referred to as mesenchymal stem cells (MSCs) or derivatives thereof. Myosin and matrix elasticity greatly affect MSC function, and these mechanobiological factors are now being explored with HSC/Ps both in vitro and in vivo. Also emerging is a role for the nucleus as a mechanically sensitive organelle that is semi-permeable to transcription factors which are modified for nuclear entry by cytoplasmic mechanobiological pathways. Since therapies envisioned with induced pluripotent stem cells and embryonic stem cells generally involve in vitro commitment to an adult stem cell or progenitor, a very deep understanding of stem cell mechanobiology is essential to progress with these multi-potent cells., (© 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Gene therapy has become of increasing interest in clinical neurosurgery with the completion of numerous clinical trials for Parkinson disease, Alzheimer disease, and pediatric genetic disorders. With improved understanding of the dysfunctional circuitry mediating various psychiatric disorders, deep brain stimulation for refractory psychiatric diseases is being increasingly explored in human patients. These factors are likely to facilitate development of gene therapy for psychiatric diseases. Because delivery of gene therapy agents would require the same surgical techniques currently being employed for deep brain stimulation, neurosurgeons are likely to lead the development of this field, as has occurred in other areas of clinical gene therapy for neurologic disorders. We review the current state of gene therapy for psychiatric disorders and focus specifically on particular areas of promising research that may translate into human trials for depression, drug addiction, obsessive-compulsive disorder, and schizophrenia. Issues that are relatively unique to psychiatric gene therapy are also discussed., (Copyright © 2013. Published by Elsevier Inc.)

Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Early growth response 2 (Egr2) is a zinc-finger transcription factor that acts as an important modulator of a variety of physiological processes, such as cell differentiation, proliferation and apoptosis. Here we showed that Egr2 was downregulated by bone morphogenetic protein (BMP) signaling pathways during the commitment of C3H10T1/2 stem cells to adipocyte lineage. Overexpression of Egr2 completely prevented BMP4-induced adipocyte lineage commitment of C3H10T1/2 stem cells, while simultaneously stimulating early smooth muscle-like differentiation. We also demonstrated that Egr2-induced early smooth muscle-like differentiation is transforming growth factor β1-independent. Egr2 can activate the transcription of early smooth muscle cell specific genes smooth muscle protein 22α and calponin 1. Together, the results indicated a novel role for Egr2 in repressing adipocyte lineage commitment and promoting early smooth muscle-like cell differentiation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The protective role of crocetin following cerebral contusion and its effects on the enhancement of angiogenesis in rats was investigated. A total of 60 Sprague-Dawley rats were divided into three groups (n = 20 each): crocetin therapy group (cerebral contusion treated with crocetin), cerebral trauma control group (without treatment), sham operation control group. The effect of crocetin was examined by modified Neurological Severity Scores (mNSS), electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) procedure, western blotting analysis of Bcl-2 protein expression, microvessel count (MVC), endothelial cell culture and immunocyto-chemistry. The mNSS results indicated that neurological function of therapy group was significantly recovered seven days and fifteen days after the trauma. The TUNEL staining and electron microscopy revealed that crocetin treatment led to an inhibition of neuronal apoptosis 72 h following treatment; this finding was confirmed by western blot analysis of B cell lymphoma/leukemia-2 (Bcl-2) protein expression. Expression levels of vascular endothelial growth factor receptor-2 (VEGFR-2) and serum response factor (SRF) were higher in the crocetin therapy group in comparison to the two other experimental groups. Our results demonstrate that the protective effects of crocetin upon brain injury may be related to its ability to inhibit apoptosis at early stages of the injury and its ability to promote angiogenesis at the sub-acute stage., (Copyright © 2011. Published by Elsevier B.V.)

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., (Copyright 2001 Academic Press.)

Serum has been known to stimulate collagen production by dermal fibroblasts. As part of an ongoing study of the molecular mechanisms of collagen production, we have investigated transcriptional regulation of the human alpha2(I) collagen gene by serum in human dermal fibroblasts. Serum responsive elements were mapped by deletion analysis between bp -353 and -264, and between -148 and -108 in the alpha2(I) collagen promoter. Further functional analysis of the alpha2(I) collagen promoter containing various substitution mutations revealed that serum stimulation of this promoter is mediated equally by a GC-rich region located between bp -303 and -271 and by the TCCTCC motif located between bp -123 and -128, both of which constitute binding sites for transcription factor Spl and Sp3. No differences were observed in electrophoretic mobility shift assays between unstimulated and serum stimulated fibroblasts. The Spl inhibitor mithramycin blocked stimulation of the alpha2(I) collagen promoter activity by serum. Furthermore, immunoprecipitation analysis showed that serum stimulation increased Spl phosphorylation. In conclusion, this study characterized response elements that mediate serum stimulation of the human alpha2(I) collagen promoter and suggests that serum stimulation was mediated via Sp1/Sp3 binding sites in this promoter.

srfA displays a complex temporal and cell type-specific pattern of expression in Dictyostelium and is expressed by most of its cell types at some stage of their development. This complexity is achieved by the use of alternative promoters. The promoter activity of the proximal region was found to be restricted to a subset of prestalk cells. Little or no associated expression was observed in the lower cup and basal disc during culmination. The middle promoter region was preferentially active in prestalk cells under usual conditions of filter development. Interestingly, during slug migration, the activity of this promoter in posterior prespore cells was strongly induced. The distal region displayed a dual pattern of expression. Thus, before culmination, this region drove lacZ expression in a few cells scattered along the entire structure. However, intense lacZ staining was found in the spores by the end of culmination. We have previously reported that srfA expression is essential for spore differentiation (R. Escalante and L. Sastre, Development 125, 3801-3808). Our novel finding of the expression of the gene in prestalk cells before culmination suggested that it might play additional roles in Dictyostelium development. The study of knockout strains revealed that srfA is also required for proper slug migration. Spore differentiation and slug migration defects were rescued by reexpression of srfA in the null mutant background, under the appropriate promoter control. The expression of srfA under the activity of the distal promoter region was able to rescue spore differentiation but not slug migration. Conversely, reexpression under the control of the middle promoter rescued slug morphogenesis and migration. Our results demonstrate that the correct spatial and temporal pattern of expression of srfA is essential for the different functions that this transcription factor plays in development., (Copyright 2001 Academic Press.)

The desmin gene encodes an intermediate filament protein that is present in skeletal, cardiac, and smooth muscle cells. This study shows that the 4-kb upstream region of the murine desmin promoter directs expression of a lacZ reporter gene throughout the heart from E7.5 and in skeletal muscle and vascular smooth muscle cells from E9. 5. The distal fragment (-4005/-2495) is active in arterial smooth muscle cells but not in venous smooth muscle cells or in the heart in vivo. It contains a CArG/octamer overlapping element (designated CArG4) that can bind the serum response factor (SRF) and an Oct-like factor. The desmin distal fragment can replace a SM22alpha regulatory region (-445/-126) that contains two CArG boxes, to cis-activate a minimal (-125/+65) SM22alpha promoter fragment in arterial smooth muscle cells of transgenic embryos. lacZ expression was abolished when mutations were introduced into the desmin CArG4 element that abolished the binding of SRF and/or Oct-like factor. These data suggest that a new type of combined CArG/octamer element plays a prominent role in the regulation of the desmin gene in arterial smooth muscle cells, and SRF and Oct-like factor could cooperate to drive specific expression in these cells., (Copyright 2000 Academic Press.)

Complementary DNA clones have been isolated from the crustacean Artemia franciscana coding for a serum response factor (SRF)-homologue that is more than 96% identical to human and Drosophila melanogaster SRFs in their MADS boxes. The SRF homologue is expressed in ectodermal tissues, as determined by in situ hybridization experiments. A SRF-binding site has been identified in the promoter region of the Actin403 gene that is also expressed in ectodermal tissues, in accordance with its transcriptional regulation by the SRF homologue. The mRNA coding for A. franciscana SRF is present at similar levels in cryptobiotic encysted embryos and in developing nauplii. However, there is a significant increase in CArG-binding activity at the later developmental stage, indicating a postranscriptional regulation of SRF during A. franciscana embryonic development.

The Drosophila serum response factor (DSRF) is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the DSRF regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signalling pathway, is sufficient to induce DSRF expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signalling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory.

This work deals with annealing of single-stranded DNA and the binding of a serum respond factor to a DNA probe containing specific binding site. Capillary electrophoresis (CE) method is explored and compared with the mobility-shift gel electrophoresis (GE) procedure. The results indicate the CE method offers direct and rapid annealing of the DNA strands. It requires no prior incubation with additives (polynucleotides, proteins) to reduce nonspecific DNA-protein interactions. Unwanted nonspecific interactions are not observed in the CE method. The presence of a fluorescein tag to the DNA probe yields identical results to those with the radioactive label. A fluorescein tag in the CE work can be used without any adverse effects. The dissociation constant (Kd) of this protein-DNA complex by the CE method was similar to those determined by the GE method (approximately 10(-6) M). The proposed method is extremely powerful, highly sensitive, quantitative, and fast. It can determine even very small conformational differences of the DNA probe.

We characterized the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in homogenates and nuclear extracts of skeletal muscle and analyzed their capacity to phosphorylate the myogenic factor SRF. Isoforms of CaMKII enriched from skeletal muscle phosphorylated SRF in vitro to high stoichiometries and produced multiple forms on SDS-PAGE, suggesting that SRF was phosphorylated at multiple sites. Phosphopeptide-mapping experiments using truncated SRF proteins located the residues of SRF phosphorylated by recombinant CaMKII within amino acids 1-171, with at least one site residing in amino acids 142-171. Microsequencing of these phosphorylated peptides identified that both Ser-103 and a novel residue, Thr-160 in the MADS box of SRF, were sites of phosphorylation. CaMKII activity was enriched in nuclear extracts relative to crude homogenates from skeletal muscle and similarly phosphorylated the nuclear transcription factor SRF in vitro. The location of Thr-160 in the 3-D structure of SRF suggests that its phosphorylation by nuclear CaMKII may directly influence DNA binding of SRF and other MADS box factors., (Copyright 2000 Academic Press.)

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors bind to and activate transcription through A+T-rich DNA sequences found primarily, but not exclusively, in the promoters of muscle-specific genes. Their importance has been established for myogenic development and in activation of the immediate-early gene, c-jun, and recently further functional roles in the immune system have emerged. The MEF2 factors belong to the MADS-box superfamily, sharing homology in a 58 amino acid domain that mediates DNA binding and dimerization. The structures of two MADS-box proteins, SRF and MCM1, bound to their cognate DNA have been previously reported and shown to share extensive similarity in their mode of DNA binding. We have solved the structure of MEF2A 2-78 bound to its DNA consensus sequence at 1.5 A resolution. It reveals how the absence of amino acids N-terminal to the MADS-box contributes to the DNA binding properties of MEF2 proteins and shows that the MEF domain C-terminal to the MADS-box adopts a conformation considerably different from the same region in SRF and MCM1., (Copyright 2000 Academic Press.)

We previously described a 110-kDa tyrosine phosphoprotein, Sob 1, that regulates formation of the DNA binding complex Band A at the c-fos serum response element (SRE) during T cell activation. Using competition and mutant oligonucleotide analysis, we have determined that both the core CArG box of the c-fos SRE and the 3' sequences flanking the CArG box are necessary for stable Band A complex formation. Moreover, using transient transfection and reporter assays, we show that mutations affecting Band A complex formation in vitro also impaired serum induction of c-fos gene expression in vivo. Since mutation at this site has no effect on SRF binding, our results suggest that in combination with SRE/SRF, Sob 1-regulated factor(s) bind at the 3' side of SRE to form Band A, and this confers maximal serum induction of c-fos gene expression via the SRE., (Copyright 1999 Academic Press.)

Transcription factor-induced DNA bending is important in determining local promoter architecture and it is thought to be a key determinant of their function. The human MADS-box transcription factors serum response factor and MEF2A exhibit different propensities to bend their binding sites. Here, we have investigated the ability of several family members from different species to bend DNA and the molecular mechanisms underlying this process. Differential DNA bending is observed in yeast and plant MADS-box proteins. Like MEF2A, the yeast proteins Rlm1 and Smp1 exhibit low DNA bending propensities. A comparison of serum response factor and SQUA reveals that the basic mechanisms of DNA bending appear to be conserved between these proteins, although several key differences do exist. In contrast to serum response factor, SQUA bends DNA in a DNA sequence-dependent manner. In both proteins, protein-DNA contacts made between residues in the beta-loop and the N-terminal end of the recognition helices in the MADS-box are the major determinants of DNA bending. However, although residues which are involved in DNA bending are predicted to be located in similar positions in their tertiary structures, different residues dictate bending by each protein. Further complexities are uncovered in the links between the DNA bending propensity and the binding specificity. In combination with structural studies, our results provide a model to explain how differential bending by MADS-box proteins is achieved at the molecular level and provide insights into how this might affect their biological function., (Copyright 1999 Academic Press.)

Serum response factor (SRF) was identified as an activity binding upon serum stimulation of HeLa cells to a motif known as the serum response element in the c-fos promoter. This element is also found in the regulatory regions of many muscle-specific genes. We have characterized srf expression during early zebrafish embryogenesis. In addition to low-level expression in many or even all cells, elevated levels of srf RNA and protein are transiently expressed in skeletal muscle lineages during their differentiation.

Cell migration and proliferation that follows injury to the artery wall is preceded by signaling and transcriptional events that converge at the promoters of multiple genes whose products can influence formation of the neointima. Transcription factors, such as early growth response factor-1 (Egr-1), with nucleotide recognition elements in the promoters of many pathophysiologically relevant genes, are expressed at the endothelial wound edge within minutes of injury. The mechanisms underlying the inducible expression of Egr-1 in this setting are not clear. Understanding this process would provide important mechanistic insights into the earliest events in the response to injury. In this report, we demonstrate that fibroblast growth factor-2 (FGF-2) is released by injury and that antibodies to FGF-2 almost completely abrogate the activation and nuclear accumulation of Egr-1. FGF-2-inducible egr-1-promoter-dependent expression is blocked by PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)-1/2 (MEK-1/2), as well as by dominant negative mutants of ERK-1/2. Inducible ERK phosphorylation after injury is dependent on release and stimulation by endogenous FGF-2. Antisense oligonucleotides directed at egr-1 mRNA suggest that Egr-1 plays a necessary role in endothelial repair after denudation of the monolayer. These findings demonstrate that inducible Egr-1 expression after injury is contingent on the release and paracrine action of FGF-2.

The activity of exogenous phosphatidic acid (PA) to transactivate c-fos serum response element (SRE) was investigated by transient transfection analysis. Incubation of Rat-2 fibroblast cells with exogenous PA caused a stimulation of c-fos SRE-linked luciferase activity in a dose- and time-dependent manner. The SRE stimulation by PA was dramatically reduced by either pre-treatment with mepacrine, an inhibitor of phospholipase A2 (PLA2), or co-transfection with antisense cytosolic phospholipase A2 (cPLA2) oligonucleotide, whereas lysophosphatidic acid (LPA)-induced SRE activation was not affected. Consistent with this specific requirement for PLA2 by PA, the translocation of cPLA2 protein was rapidly induced followed by PA treatment. Together, these results suggest that PLA2, especially cPLA2, plays a critical role in the nuclear signaling cascade of PA in Rat-2 fibroblast cells.

Cultured mesangial cells constitutively express alpha-smooth muscle actin (alpha-SMA), a marker of cellular activation. We unexpectedly found that tyrosine kinase pp60v-src, a known activator for a wide range of signalling cascades, suppressed the alpha-SMA expression in mesangial cells. The present study was conducted to elucidate molecular events involved in this phenomenon. Transfection with a reporter plasmid revealed that the serum response element (SRE), the cis-element required for alpha-SMA expression, was constitutively active in mesangial cells. When mesangial cells were transfected with pp60v-src, activity of both SRE and the alpha-SMA promoter was down-regulated. This was associated with depressed levels of phosphorylated extracellular signal-regulated kinases (ERKs), but not c-Jun N-terminal kinase. Selective inhibition of ERKs by PD098059 abrogated constitutive SRE activity, leading to suppressed alpha-SMA expression. These results uncovered a novel potential of pp60v-src for suppression of alpha-SMA via intervention in the ERK-SRE signalling pathway.

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 multiple cis/trans interactions found along the smooth muscle gamma-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 products in 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., (Copyright 1998 Academic Press.)

A 22-bp fragment including the CArG element (CArG1) is essential for the transcription of the caldesmon gene. In this study, we investigated the effects of serum response factor (SRF) on the functional regulation of caldesmon promoter in smooth muscle cells. Gel supershift assay revealed that SRF was one component of the CArG1-protein complex. Dominant-negative mutants of SRF suppressed the promoter activity of caldesmon, whereas wild-type SRF overcame this suppression. These results suggest that SRF functions as a core activating factor of the caldesmon promoter. Furthermore, fractionation of smooth muscle cells' nuclear extracts using DNA affinity paramagnetic particles suggests that SRF transactivates the caldesmon promoter in concert with additional factors in the flow-through fraction recruited to the CArG element.

v-Src transcriptionally induces gene expression by activating several transcriptional response elements such as the serum response element (SRE), the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE), and the c-AMP response element (CRE) found in the promoters of several proliferation-related immediate early genes. We report here that a Src protein, with a deletion in the SH3 domain and lacking a membrane localization signal, strongly activates gene expression mediated by SRE, TRE and CRE transcriptional control elements. This mutant was unable to cause cellular transformation, suggesting that activation of these transcriptional control elements is not sufficient for the induction of a transformed phenotype by Src. Interestingly, the ability of the membrane localization and SH3 deletion mutant to activate gene expression was abolished upon conversion of the C-terminal inhibitory Tyr-527 to Phe. These data suggest the existence of previously unreported Tyr-527-dependent activation of intracellular signals that activate gene expression. These data raise the possibility that Src may exert physiological effects via an interaction between Tyr-527 and an SH2-containing protein that would interact with the phosphorylated form of Tyr-527.

SM22alpha is an adult smooth muscle-specific protein that is expressed in the smooth, cardiac, and skeletal muscle lineages during early embryogenesis before becoming restricted specifically to all vascular and visceral smooth muscle cells (SMC) in late fetal development and adulthood. We have used the SM22alpha gene as a marker to define the regulatory mechanisms that control muscle-specific gene expression in SMCs. Previously, we reported that the 445-base-pair promoter of SM22alpha was sufficient to direct transcription of a lacZ reporter gene in early cardiac and skeletal muscle cell lineages and in a subset of arterial SMCs, but not in venous nor visceral SMCs in transgenic mice. Here we describe two evolutionarily conserved CArG (CC(A/T)6GG) boxes in the SM22alpha promoter, both of which are essential for full promoter activity in cultured SMCs. In contrast, only the promoter-proximal CArG box is essential for specific expression in developing smooth, skeletal, and cardiac muscle lineages in transgenic mice. Both CArG boxes bind serum response factor (SRF), but SRF binding is not sufficient for SM22alpha promoter activity, since overexpression of SRF in the embryonal teratocarcinoma cell line F9, which normally expresses low levels of SRF, fails to activate the promoter. However, a chimeric protein in which SRF was fused to the transcription activation domain of the viral coactivator VP16 is able to activate the SM22alpha promoter in F9 cells. These results demonstrate the SM22alpha promoter-proximal CArG box is a target for the regulatory programs that confer smooth, skeletal, and cardiac muscle specificity to the SM22alpha promoter and they suggest that SRF activates SM22alpha transcription in conjunction with additional regulatory factors that are cell type-restricted.

To identify the functional regulatory elements of the promoter of the cytoplasmic actin A3 gene in Bombyx mori, transient expression of A3-LacZ mutants was assayed in cultured Lepidoptera cells. This led to the recognition of two proximal and contiguous domains exerting strong negative and positive effects, respectively on promoter activity. The negative region contains a ten-base-pair sequence that binds Bombyx silk gland cell nuclear proteins in vitro. The positive regulatory element was identified as a serum response element (SRE) by its sequence, and its in vitro binding properties. Moreover, structural analysis of posterior and median silk gland cell chromatin by dimethyl sulfate-aided LMPCR revealed that SRE is bound to its cognate factor in situ, in most, if not all, the approximately 100,000 A3 copies of the polyploid DNA stock. The regulation of the A3 promoter in the silk gland would thus result from the combined action of these two antagonist factors.