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1.
Sci Rep ; 10(1): 5281, 2020 03 24.
Article in English | MEDLINE | ID: mdl-32210317

ABSTRACT

Traumatic injury of peripheral nerves typically also damages nerve surrounding tissue including muscles. Hence, molecular and cellular interactions of neighboring damaged tissues might be decisive for successful axonal regeneration of injured nerves. So far, the contribution of muscles and muscle-derived molecules to peripheral nerve regeneration has only poorly been studied. Herein, we conditionally ablated SRF (serum response factor), an important myofiber transcription factor, in skeletal muscles of mice. Subsequently, the impact of this myofiber-restricted SRF deletion on peripheral nerve regeneration, i.e. facial nerve injury was analyzed. Quantification of facial nerve regeneration by retrograde tracer transport, inspection of neuromuscular junctions (NMJs) and recovery of whisker movement revealed reduced axonal regeneration upon muscle specific Srf deletion. In contrast, responses in brainstem facial motor neuron cell bodies such as regeneration-associated gene (RAG) induction of Atf3, synaptic stripping and neuroinflammation were not overly affected by SRF deficiency. Mechanistically, SRF in myofibers appears to stimulate nerve regeneration through regulation of muscular satellite cell (SC) proliferation. In summary, our data suggest a role of muscle cells and SRF expression within muscles for regeneration of injured peripheral nerves.


Subject(s)
Facial Muscles/metabolism , Facial Nerve Injuries/physiopathology , Facial Nerve/physiology , Masseter Muscle/metabolism , Nerve Regeneration/physiology , Serum Response Factor/physiology , Activating Transcription Factor 3/biosynthesis , Activating Transcription Factor 3/genetics , Animals , Brain Stem/physiopathology , Facial Muscles/innervation , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Lip/innervation , Masseter Muscle/innervation , Mice , Motor Neurons/physiology , Organ Specificity , Promoter Regions, Genetic , Recombinant Fusion Proteins/metabolism , Satellite Cells, Skeletal Muscle/physiology , Serum Response Factor/biosynthesis , Serum Response Factor/deficiency , Serum Response Factor/genetics , Up-Regulation , Vibrissae/innervation
2.
Circ Res ; 126(1): 41-56, 2020 01 03.
Article in English | MEDLINE | ID: mdl-31656131

ABSTRACT

RATIONALE: The mitochondrial Poldip2 (protein polymerase interacting protein 2) is required for the activity of the tricarboxylic acid cycle. As a consequence, Poldip2 deficiency induces metabolic reprograming with repressed mitochondrial respiration and increased glycolytic activity. Though homozygous deletion of Poldip2 is lethal, heterozygous mice are viable and show protection against aneurysm and injury-induced neointimal hyperplasia, diseases linked to loss of vascular smooth muscle differentiation. Thus, we hypothesize that the metabolic reprograming induced by Poldip2 deficiency controls VSMC differentiation. OBJECTIVE: To determine the role of Poldip2-mediated metabolic reprograming in phenotypic modulation of VSMC. METHODS AND RESULTS: We show that Poldip2 deficiency in vascular smooth muscle in vitro and in vivo induces the expression of the SRF (serum response factor), myocardin, and MRTFA (myocardin-related transcription factor A) and dramatically represses KLF4 (Krüppel-like factor 4). Consequently, Poldip2-deficient VSMC and mouse aorta express high levels of contractile proteins and, more significantly, these cells do not dedifferentiate nor acquire macrophage-like characteristics when exposed to cholesterol or PDGF (platelet-derived growth factor). Regarding the mechanism, we found that Poldip2 deficiency upregulates the hexosamine biosynthetic pathway and OGT (O-linked N-acetylglucosamine transferase)-mediated protein O-GlcNAcylation. Increased protein glycosylation causes the inhibition of a nuclear ubiquitin proteasome system responsible for SRF stabilization and KLF4 repression and is required for the establishment of the differentiated phenotype in Poldip2-deficient cells. CONCLUSIONS: Our data show that Poldip2 deficiency induces a highly differentiated phenotype in VSMCs through a mechanism that involves regulation of metabolism and proteostasis. Additionally, our study positions mitochondria-initiated signaling as key element of the VSMC differentiation programs that can be targeted to modulate VSMC phenotype during vascular diseases.


Subject(s)
Mitochondrial Proteins/physiology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Nuclear Proteins/physiology , Animals , Cell Differentiation , Cells, Cultured , Gene Expression Regulation , Humans , Hyperplasia , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Myocytes, Smooth Muscle/cytology , Neointima , Nuclear Proteins/biosynthesis , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Phenotype , Proteasome Endopeptidase Complex/metabolism , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Trans-Activators/biosynthesis , Trans-Activators/genetics , Ubiquitin/metabolism
3.
Prostate ; 78(10): 724-730, 2018 07.
Article in English | MEDLINE | ID: mdl-29608018

ABSTRACT

BACKGROUND: Up to a third of prostate cancer patients fail curative treatment strategies such as surgery and radiation therapy in the form of biochemical recurrence (BCR) which can be predictive of poor outcome. Recent clinical trials have shown that men experiencing BCR might benefit from earlier intervention post-radical prostatectomy (RP). Therefore, there is an urgent need to identify earlier prognostic biomarkers which will guide clinicians in making accurate diagnosis and timely decisions on the next appropriate treatment. The objective of this study was to evaluate Serum Response Factor (SRF) protein expression following RP and to investigate its association with BCR. MATERIALS AND METHODS: SRF nuclear expression was evaluated by immunohistochemistry (IHC) in TMAs across three international radical prostatectomy cohorts for a total of 615 patients. Log-rank test and Kaplan-Meier analyses were used for BCR comparisons. Stepwise backwards elimination proportional hazard regression analysis was used to explore the significance of SRF in predicting BCR in the context of other clinical pathological variables. Area under the curve (AUC) values were generated by simulating repeated random sub-samples. RESULTS: Analysis of the immunohistochemical staining of benign versus cancer cores showed higher expression of nuclear SRF protein expression in cancer cores compared with benign for all the three TMAs analysed (P < 0.001, n = 615). Kaplan-Meier curves of the three TMAs combined showed that patients with higher SRF nuclear expression had a shorter time to BCR compared with patients with lower SRF expression (P < 0.001, n = 215). Together with pathological T stage T3, SRF was identified as a predictor of BCR using stepwise backwards elimination proportional hazard regression analysis (P = 0.0521). Moreover ROC curves and AUC values showed that SRF was better than T stage in predicting BCR at year 3 and 5 following radical prostatectomy, the combination of SRF and T stage had a higher AUC value than the two taken separately. CONCLUSIONS: SRF assessment by IHC following RP could be useful in guiding clinicians to better identify patients for appropriate follow-up and timely treatment.


Subject(s)
Neoplasm Recurrence, Local/metabolism , Prostate/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/surgery , Serum Response Factor/biosynthesis , Aged , Humans , Immunochemistry , Male , Middle Aged , Neoplasm Recurrence, Local/blood , Neoplasm Recurrence, Local/pathology , Prostate/surgery , Prostatic Neoplasms/blood , Prostatic Neoplasms/pathology , Serum Response Factor/blood , Survival Analysis
4.
Alcohol Clin Exp Res ; 39(10): 1951-6, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26342644

ABSTRACT

BACKGROUND: Deficits in neuronal plasticity underlie many neurobehavioral and cognitive problems presented in fetal alcohol spectrum disorder (FASD). Our laboratory has developed a ferret model showing that early alcohol exposure leads to a persistent disruption in ocular dominance plasticity (ODP). For instance, a few days of monocular deprivation results in a robust reduction of visual cortex neurons' responsiveness to stimulation of the deprived eye in normal animals, but not in ferrets with early alcohol exposure. Previously our laboratory demonstrated that overexpression of serum response factor (SRF) exclusively in astrocytes can improve neuronal plasticity in FASD. Here, we test whether neuronal overexpression of SRF can achieve similar effects. METHODS: Ferrets received 3.5 g/kg alcohol intraperitoneally (25% in saline) or saline as control every other day between postnatal day 10 to 30, which is roughly equivalent to the third trimester of human gestation. Animals were given intracortical injections of a Herpes Simplex Virus-based vector to express either green fluorescent protein or a constitutively active form of SRF in infected neurons. They were then monocularly deprived by eyelid suture for 4 to 5 days after which single-unit recordings were conducted to determine whether changes in ocular dominance had occurred. RESULTS: Overexpression of a constitutively active form of SRF by neurons restored ODP in alcohol-treated animals. This effect was observed only in areas near the site of viral infection. CONCLUSIONS: Overexpression of SRF in neurons can restore plasticity in the ferret model of FASD, but only in areas near the site of infection. This contrasts with SRF overexpression in astrocytes which restored plasticity throughout the visual cortex.


Subject(s)
Dominance, Ocular/physiology , Ethanol/adverse effects , Ferrets , Fetal Alcohol Spectrum Disorders/physiopathology , Neuronal Plasticity/physiology , Neurons/metabolism , Serum Response Factor/biosynthesis , Animals , Animals, Genetically Modified , Disease Models, Animal , Fetal Alcohol Spectrum Disorders/metabolism , Visual Cortex/physiopathology
5.
Am J Physiol Lung Cell Mol Physiol ; 309(1): L37-45, 2015 Jul 01.
Article in English | MEDLINE | ID: mdl-25979077

ABSTRACT

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors.


Subject(s)
Asthma/pathology , Horse Diseases/pathology , Nuclear Proteins/biosynthesis , Serum Response Factor/biosynthesis , Trans-Activators/biosynthesis , ets-Domain Protein Elk-1/biosynthesis , Airway Remodeling/immunology , Animals , Asthma/immunology , Asthma/metabolism , Cell Nucleus/metabolism , Cell Proliferation , Disease Models, Animal , Horse Diseases/immunology , Horse Diseases/metabolism , Horses , Hyperplasia/pathology , Hypertrophy/pathology , Muscle Contraction , Muscle, Smooth/metabolism , Nuclear Proteins/metabolism , Protein Binding , Trans-Activators/metabolism
6.
J Cell Biochem ; 116(10): 2166-76, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25639779

ABSTRACT

Cardiac hypertrophy is the response of the heart to a variety of hypertrophic stimuli; this condition progresses to heart failure and sudden death. MicroRNAs (miRs) are a family of small, non-coding RNAs that mediate posttranscriptional gene silencing. Recent studies have identified miRs as important regulators in cardiac hypertrophy. One specific miR, miR-150 has been reported to be downregulated in hypertrophic murine hearts. However, the role of miR-150 as a regulator of cardiac hypertrophy remains unclear. In the present study, we used gain-of-function and loss-of-function approaches to investigate the functional roles of miR-150 in cardiac hypertrophy induced by aortic banding. The extent of the cardiac hypertrophy was evaluated by echocardiography and by pathological and molecular analyses of heart samples. Our results revealed that transgenic mice that overexpress miR-150 in the heart were resistant to cardiac hypertrophy and fibrosis through down-regulation of serum response factor (SRF). Conversely, the loss of function of miR-150 by genetic knockdown or antagomiR approaches produced the opposite effects. These studies suggest that miR-150 plays an important role in the regulation of cardiac hypertrophy and SRF is involved in miR-150 mediated anti-hypertrophic effect. Thus, miR-150 may be a new therapeutic target for cardiac hypertrophy.


Subject(s)
Arterial Pressure/genetics , Cardiomegaly/genetics , MicroRNAs/genetics , Serum Response Factor/biosynthesis , Animals , Cardiomegaly/pathology , Disease Models, Animal , Echocardiography , Fibrosis/genetics , Fibrosis/pathology , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Mice , Mice, Transgenic , MicroRNAs/biosynthesis , Serum Response Factor/genetics
7.
Glia ; 60(12): 1906-14, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22907787

ABSTRACT

Serum response factor (SRF) is a transcription factor that transactivates actin-associated genes and has been implicated in oligodendrocyte (OL) differentiation. To date, it has not been investigated in cerebral ischemia. We investigated the dynamics of SRF expression after stroke in vivo and the role of SRF in OL differentiation in vitro. Using immunohistochemistry, we found that SRF was upregulated in OLs and OL precursor cells (OPCs) after stroke. Moreover, upregulation of SRF was concurrent with downregulation of the micro-RNAs (miRNAs) miR-9 and the miR-200 family in the ischemic white matter region, the corpus callosum. Inhibition of SRF activation by CCG-1423, a specific inhibitor of SRF function, blocked OPCs from differentiating into OLs. Overexpression of miR-9 and miR-200 in cultured OPCs suppressed SRF expression and inhibited OPC differentiation. Moreover, co-expression of miR-9 and miR-200 attenuated activity of a luciferase reporter assay containing the Srf 3' untranslated region. Collectively, this study is the first to show that stroke upregulates SRF expression in OPCs and OLs, and that SRF levels are mediated by miRNAs and regulate OPC differentiation.


Subject(s)
Cell Differentiation/physiology , MicroRNAs/physiology , Oligodendroglia/physiology , Serum Response Factor/biosynthesis , Stem Cells/physiology , Animals , Cells, Cultured , Male , MicroRNAs/antagonists & inhibitors , Oligodendroglia/cytology , Rats , Rats, Wistar , Serum Response Factor/physiology , Stem Cells/cytology , Stroke/metabolism , Stroke/pathology
8.
Thorax ; 67(1): 26-34, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21998125

ABSTRACT

RATIONALE: Muscle atrophy confers a poor prognosis in patients with chronic obstructive pulmonary disease (COPD), yet the molecular pathways responsible are poorly characterised. Muscle-specific microRNAs and serum response factor (SRF) are important regulators of muscle phenotype that contribute to a feedback system to regulate muscle gene expression. The role of these factors in the skeletal muscle dysfunction that accompanies COPD is unknown. METHODS: 31 patients with COPD and 14 healthy age-matched controls underwent lung and quadriceps function assessments, measurement of daily activity and a percutaneous quadriceps muscle biopsy. The expression of muscle-specific microRNAs, myosin heavy chains and components of the serum response factor signalling pathway were determined by qPCR. RESULTS: A reduction in expression of miR-1 (2.5-fold, p=0.01) and the myocardin-related transcription factors (MRTFs) A and B was observed in patients compared with controls (MRTF-A mRNA: twofold, p=0.028; MRTF-B mRNA: fourfold, p=0.011). miR-1 expression was associated with smoking history, lung function, fat-free mass index, 6 min walk distance and percentage of type 1 fibres. miR-133 and miR-206 were negatively correlated with daily physical activity. Insulin-like growth factor 1 mRNA was increased in the patients and miR-1 was negatively correlated with phosphorylation of the kinase Akt. Furthermore, the protein levels of histone deacetylase 4, another miR-1 target, were increased in the patients. CONCLUSIONS: Downregulation of the activity of the MRTF-SRF axis and the expression of muscle-specific microRNAs, particularly miR-1, may contribute to COPD-associated skeletal muscle dysfunction.


Subject(s)
Down-Regulation , MicroRNAs/genetics , Muscular Diseases/etiology , Pulmonary Disease, Chronic Obstructive/genetics , Quadriceps Muscle/metabolism , Serum Response Factor/genetics , Aged , Biopsy , Disease Progression , Female , Fluorescent Antibody Technique , Follow-Up Studies , Genetic Predisposition to Disease , Humans , Male , MicroRNAs/biosynthesis , Muscular Diseases/genetics , Muscular Diseases/metabolism , Pulmonary Disease, Chronic Obstructive/complications , Pulmonary Disease, Chronic Obstructive/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serum Response Factor/biosynthesis , Signal Transduction
9.
Mol Cell Biol ; 32(3): 633-51, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22144583

ABSTRACT

MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor ß (TGF-ß)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.


Subject(s)
Breast Neoplasms/pathology , Fetal Proteins/metabolism , MicroRNAs/metabolism , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Breast Neoplasms/metabolism , Cardiac Myosins/biosynthesis , Cell Line, Tumor , Cell Movement , DNA-Binding Proteins/metabolism , Down-Regulation , Female , Formins , Gene Expression Regulation, Neoplastic , Humans , Myosin Light Chains/biosynthesis , Neoplasm Invasiveness , Oncogene Proteins, Fusion/metabolism , Serum Response Factor/biosynthesis , Stress Fibers/metabolism , Trans-Activators , Transforming Growth Factor beta/metabolism
10.
J Ethnopharmacol ; 117(3): 473-7, 2008 May 22.
Article in English | MEDLINE | ID: mdl-18417308

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: ShuXueTong injection is a traditional Chinese drug designed to treat the patients of "blood stasis and stagnation (yu xue yu zhi)", including subacute brain trauma. However, the mechanism of the therapeutic effect of ShuXueTong on traumatic brain injury is unknown yet. AIM OF THE STUDY: We hypothesized that ShuXueTong may promote the brain wound healing by facilitating angiogenesis. Thus this study was designed to explore this hypothesis. MATERIALS AND METHODS: By means of microvessel count, Western blotting, immunocytochemistry, methyl thiazolyl tetrazolium assay and etc., the effect of ShuXueTong on the angiogenesis of brain wound was studied and then its influence on the VEGF/VEGFR-2 pathway were explored. RESULTS: ShuXueTong facilitates angiogenesis in the brain wound and improves the neurological function of the traumatized rats. VEGF expression in the lesion was elevated due to ShuXueTong induction. The in vitro experiment revealed VEGFR-2 and SRF expression in the endothelial cells were enhanced when exposed to ShuXueTong for merely 1d. Moreover, ShuXueTong promoted the endothelial cell proliferation via the VEGF/VEGFR-2 pathway. CONCLUSIONS: The mechanism of the therapeutic effect of ShuXueTong on traumatic brain injury lies at least partly in the enhanced angiogenesis in the lesion.


Subject(s)
Angiogenesis Modulating Agents/pharmacology , Brain Injuries/drug therapy , Drugs, Chinese Herbal/pharmacology , Neovascularization, Physiologic/drug effects , Wound Healing/drug effects , Animals , Brain Injuries/pathology , Capillaries/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Male , Medicine, Chinese Traditional , Motor Activity/physiology , Paralysis/drug therapy , Paralysis/etiology , Rats , Rats, Sprague-Dawley , Receptors, Vascular Endothelial Growth Factor/metabolism , Serum Response Factor/biosynthesis , Tetrazolium Salts , Thiazoles , Vascular Endothelial Growth Factor A/biosynthesis
11.
Basic Res Cardiol ; 102(3): 233-44, 2007 May.
Article in English | MEDLINE | ID: mdl-17122890

ABSTRACT

To understand the effect of transcription regulation in modulating cardiac aging, we sought to study the role of serum response factor (SRF), a key transcription factor in the heart that is normally increased with senescence and also in congestive heart failure. A Tet-Off gene expression system was used for cardiac-specific over-expression of a mutant SRF protein. In these binary transgenic mice, there is no age-related increase in SRF protein expression; in fact, there appeared to be a mild reduction of SRF protein (Mild-R SRF Tg). The older, middle-aged (15 mo) Mild-R SRF Tg mice appeared healthier and were better able to maintain their left ventricular systolic pressure (LVSP) in response to moderate â-adrenergic stimulation compared with age-matched Non-Tg mice, which demonstrated a negative ionotropic response. The Mild-R SRF Tg hearts had lower mRNA expression of BNP (p < 0.05), and the sodium calcium exchanger (p < 0.05), compared to Non-Tg. Mild-R SRF Tg had higher mRNA levels of SERCA2 (p < 0.05) and ryanodine receptor 2 (p < 0.05) compared to Non-Tg hearts. These findings suggest that preventing the age-associated increase in SRF is associated with better preserved intracellular calcium handling and functional response to stress; it might be advantageous for the older adult heart. This mouse model could be helpful in elucidating the molecular mechanisms underlying certain age-related changes in cardiac reserve capacity and response to stress.


Subject(s)
Heart/drug effects , Isoproterenol/pharmacology , Serum Response Factor/biosynthesis , Ventricular Pressure/drug effects , Age Factors , Animals , Blotting, Western , Disease Models, Animal , Gene Expression Regulation , Heart/physiopathology , Mice , Mice, Transgenic , Myocardium/pathology , Reverse Transcriptase Polymerase Chain Reaction , Serum Response Factor/analysis
12.
Toxicol Sci ; 96(1): 83-91, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17116645

ABSTRACT

The transcriptional activator serum response factor (SRF) is a member of the immediate early gene family known to promote embryonic development, cell growth, and myogenesis through interaction with multiple nuclear protein factors. Previous studies have shown that SRF possesses potent transcriptional activation domains that can interfere with gene expression at artificially high expression levels through "transcriptional squelching." The current work sought to characterize toxicological aspects of SRF-mediated transcriptional squelching. An adenoviral expression system driven by the potent cytomegalovirus promoter was used to achieve up to a 50-fold increase in SRF protein levels. The overexpressed SRF is nuclear localized and interferes with gene expression independent of specific promoter interaction as expected for transcriptional squelching. SRF-mediated squelching elicits robust cell killing affecting multiple cell types including normal and abnormal proliferating cells as well as postmitotic cells such as cardiomyocytes in culture, and the cell killing is more pronounced than that mediated by the tumor suppressor protein p53. Although both the DNA-binding and transcriptional activation domains of SRF are normally required for the physiological roles of SRF, only the transcriptional activation domain is required for cell killing. Unlike c-myc-induced cell killing, squelching-induced cell death does not require serum withdrawal and cannot be effectively attenuated by blocking the caspase and calpain proteolytic pathways or by overexpression of the antiapoptotic gene bcl-xL. These findings suggest transcriptional squelching may be engineered for killing cancer cells, and the SRF gene may represent a novel molecular target for cancer therapeutics.


Subject(s)
Cell Nucleus/metabolism , Serum Response Factor/biosynthesis , Transcription, Genetic , Transcriptional Activation , Adenoviridae/genetics , Animals , Cell Death/genetics , Cell Line , Cell Survival/genetics , Genetic Vectors , Humans , Mutation , Protein Binding , Serum Response Element/genetics , Serum Response Factor/genetics , Time Factors , Transfection
13.
J Neurosci ; 26(39): 10020-32, 2006 Sep 27.
Article in English | MEDLINE | ID: mdl-17005865

ABSTRACT

Serum response factor (SRF)-mediated transcription contributes to developmental and adult brain plasticity. Therefore, we investigated the role of a newly identified SRF coactivator, MKL1, in the regulation of SRF-driven transcription in rat forebrain neurons. MKL1 expression was found in newborn rat cortical or hippocampal neurons in culture as well as in adult rat forebrain. Immunostaining demonstrated constitutive nuclear localization of MKL1 in the CA1 region of the hippocampus, in the deep layers of the neocortex, and in cultured neurons. Overexpression of MKL1 in primary cortical neurons elevated SRF-driven transcription and enhanced its stimulation by BDNF. In addition, inhibition of endogenous MKL1 by overexpression of a dominant-negative MKL1 mutant or by small interfering RNA reduced BDNF activation of SRF-driven transcription. In neurons, endogenous MKL1 was associated with SRF-regulated chromatin regions of several endogenous genes including c-fos, JunB, Srf, and Cyr61. BDNF activation of MKL1/SRF-driven transcription was dependent on the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which also led to MKL1 phosphorylation. Finally, synaptic activity stimulation of SRF-driven transcription was reduced by inhibition of endogenous MKL1. Conversely, synaptic activity enhanced transcription by overexpressed MKL1. MKL1 regulation by synaptic activity was mediated through the NMDA receptor-activated ERK1/2. These results suggest that neuronal MKL1 contributes to SRF-regulated gene expression induced by BDNF or synaptic activity. In addition, MKL1 appears as a novel mediator of the signaling between ERK1/2 and SRF. Moreover, MKL1 is a likely regulator of SRF-driven transcription programs that underlie neuronal plasticity.


Subject(s)
Brain-Derived Neurotrophic Factor/physiology , Gene Expression Regulation/physiology , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/physiology , Neurons/physiology , Serum Response Factor/physiology , Synaptic Transmission/physiology , Transcription Factors/physiology , Transcription, Genetic/physiology , 4-Aminopyridine/pharmacology , Amides/pharmacology , Animals , Animals, Newborn , Bicuculline/pharmacology , Butadienes/pharmacology , COS Cells , Cells, Cultured/metabolism , Cerebral Cortex/cytology , Chlorocebus aethiops , Chromones/pharmacology , Cysteine-Rich Protein 61 , Genes, fos , Hippocampus/cytology , Immediate-Early Proteins/biosynthesis , Immediate-Early Proteins/genetics , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Morpholines/pharmacology , N-Methylaspartate/pharmacology , Neurons/chemistry , Nitriles/pharmacology , Potassium Chloride/pharmacology , Prosencephalon/cytology , Proto-Oncogene Proteins c-fos/biosynthesis , Proto-Oncogene Proteins c-jun/biosynthesis , Proto-Oncogene Proteins c-jun/genetics , Pyramidal Cells/drug effects , Pyramidal Cells/metabolism , Pyridines/pharmacology , RNA Interference , Rats , Rats, Sprague-Dawley , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Signal Transduction/physiology , Transcription Factors/analysis , Transcription Factors/genetics , Transfection
14.
Cancer Res ; 65(21): 9883-90, 2005 Nov 01.
Article in English | MEDLINE | ID: mdl-16267012

ABSTRACT

Our initial characterization of Rac3, a close relative of the small GTPase Rac1, established its ability to promote membrane ruffling, transformation, and activation of c-jun transcriptional activity. The finding that Rac3 is transforming, and its similarity to Rac1, a protein that has a well-established connection to many processes important for cancer progression, prompted further investigation into Rac3 transformation. We used effector domain mutants (EDMs) to explore the relationship among Rac signaling, transformation, and effector usage. All Rac3 EDMs tested (N26D, F37L, Y40C, and N43D) retained the ability to promote membrane ruffling and focus formation. In contrast, only the N43D mutant promoted anchorage independence. This differs from Rac1, where both N26D and N43D mutants were impaired in both types of transformation. To learn more about the signaling pathways involved, we did luciferase reporter assays and glutathione S-transferase pull-down assays for effector binding. We found evidence for a functional link between activation of phospholipase Cbeta2 by Rac3 and signaling to the serum response factor (SRF). Surprisingly, we also found that Rac3 binds poorly to the known Rac1 effectors mixed lineage kinases 2 and 3 (MLK2 and MLK3). Transcription of cyclin D1 was the only pathway that correlated with growth in soft agar. Our experiments show that activation of membrane ruffling and transcriptional activation of c-jun, SRF, or E2F are not sufficient to promote anchorage-independent growth mediated by Rac3. Instead, multiple effector pathways are required for Rac3 transformation, and these overlap partially but not completely with those used by Rac1.


Subject(s)
Cell Transformation, Neoplastic/genetics , rac GTP-Binding Proteins/genetics , Animals , COS Cells , Carrier Proteins/metabolism , Cell Adhesion/genetics , Cell Growth Processes/genetics , Cell Transformation, Neoplastic/pathology , Chlorocebus aethiops , Isoenzymes/metabolism , Mice , NIH 3T3 Cells , Neuropeptides/metabolism , Phospholipase C beta , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Transcriptional Activation , Type C Phospholipases/metabolism , p21-Activated Kinases , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein
15.
Sheng Li Xue Bao ; 57(3): 295-302, 2005 Jun 25.
Article in Chinese | MEDLINE | ID: mdl-15968423

ABSTRACT

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.


Subject(s)
Actins/biosynthesis , Endothelium, Vascular/cytology , Serum Response Factor/biosynthesis , rhoA GTP-Binding Protein/physiology , Actins/genetics , Cytoskeleton/metabolism , Endothelium, Vascular/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Protein Serine-Threonine Kinases/metabolism , Serum Response Factor/genetics , Umbilical Veins/cytology , rho-Associated Kinases
16.
Arterioscler Thromb Vasc Biol ; 25(2): 341-7, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15591224

ABSTRACT

OBJECTIVE: Transforming growth factor-beta1 (TGF-beta1) controls the expression of numerous genes, including smooth muscle cell (SMC)-specific genes and extracellular matrix protein genes. Here we investigated whether c-Src plays a role in TGF-beta1 signaling in mouse embryonic fibroblast C3H10T1/2 cells. METHODS AND RESULTS: TGF-beta1 induction of the SMC contractile protein SM22alpha gene expression was inhibited by PP1 (an inhibitor of Src family kinases) or by C-terminal Src kinase (a negative regulator of c-Src). Induction of SM22alpha by TGF-beta1 was markedly attenuated in SYF cells (c-Src(-), Yes(-), and Fyn(-)) compared with Src(++) cells (c-Src(++), Yes(-), and Fyn(-)). PP1 also inhibited the TGF-beta1-induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-beta1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-beta1-induced expression of SM22alpha. Furthermore, we demonstrate that TGF-beta1 induction of the plasminogen activator inhibitor-1 (PAI-1) gene, which is known to be dependent on Smad but not on SRF, is inhibited by PP1 and apocynin. CONCLUSIONS: Our results suggest that TGF-beta1 activates c-Src and generates hydrogen peroxide through NAD(P)H oxidase, and these signaling pathways lead to the activation of specific sets of genes, including SM22alpha and PAI-1. TGF-beta1 controls the expression of numerous genes, including SM22alpha and PAI-1. We investigated whether c-Src plays a role in TGF-beta1 signaling. TGF-beta1 induction of such genes was significantly reduced in Src family tyrosine kinase-deficient cells, and Csk and pharmacological inhibitors for Src family kinases or antioxidants inhibit the effects of TGF-beta1. These results indicate that c-Src and hydrogen peroxide are required for TGF-beta1 signaling.


Subject(s)
Fibroblasts/drug effects , Gene Expression Regulation/physiology , Hydrogen Peroxide/metabolism , Microfilament Proteins/biosynthesis , Muscle Proteins/biosynthesis , Plasminogen Activator Inhibitor 1/biosynthesis , Proto-Oncogene Proteins pp60(c-src)/physiology , Signal Transduction/physiology , Transforming Growth Factor beta/pharmacology , Acetophenones/pharmacology , Acetylcysteine/pharmacology , Animals , Catalase/pharmacology , Cell Line/drug effects , Cell Line/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Ditiocarb/pharmacology , Fibroblasts/physiology , Flavonoids/pharmacology , Fluoresceins/pharmacology , Gene Expression Regulation/drug effects , Gene Targeting , Mice , Mice, Inbred C3H , Microfilament Proteins/genetics , Muscle Proteins/genetics , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/physiology , Naphthalenes/pharmacology , Plasminogen Activator Inhibitor 1/genetics , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Recombinant Fusion Proteins/physiology , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Signal Transduction/drug effects , Smad3 Protein , Smad4 Protein , Smad6 Protein , Sulfones/pharmacology , Trans-Activators/genetics , Trans-Activators/physiology , Transforming Growth Factor beta/physiology , Transforming Growth Factor beta1 , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/deficiency , src-Family Kinases/genetics , src-Family Kinases/physiology
17.
J Histochem Cytochem ; 52(11): 1511-8, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15505346

ABSTRACT

Intimal thickening is an early phase of atherosclerosis characterized by differentiation of plaque smooth muscle cells (SMCs) from a contractile to a synthetic phenotype. We used laser microdissection (LMD) plus real-time RT-PCR to quantify mRNAs for calponin-1 and smoothelin, markers of the contractile phenotype, and for serum response factor (SRF), a regulator of SMC differentiation, in intimal and medial SMCs of human coronary arteries with intimal thickening. RNA expression was also analyzed by ISH and protein expression was detected by IHC. LMD plus RT-PCR found similar levels of SRF mRNA in intimal and medial SMCs, while medial mRNA levels for calponin-1 and smoothelin were higher. ISH confirmed that smoothelin mRNA levels in media exceeded those in intima, whereas SRF mRNA levels were similar at both sites. For calponin-1 and smoothelin, protein levels mirrored respective mRNA levels. By contrast, more medial than intimal SRF protein was present. Our results indicate that intimal SMCs exhibit a largely synthetic phenotype, perhaps reflecting lower intimal levels of SRF protein; ISH and LMD plus real-time RT-PCR provide comparable results; as a valuable alternative to ISH, LMD plus RT-PCR allows parallel measurement of several transcripts; and tissue gene expression studies must measure both protein and mRNA levels.


Subject(s)
Coronary Vessels/metabolism , RNA, Messenger/biosynthesis , Tunica Intima/metabolism , Biomarkers , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , Coronary Vessels/pathology , Cytoskeletal Proteins/biosynthesis , Cytoskeletal Proteins/genetics , Humans , Immunohistochemistry , In Situ Hybridization , Lasers , Microfilament Proteins , Muscle Proteins/biosynthesis , Muscle Proteins/genetics , Myocytes, Smooth Muscle/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Tunica Intima/pathology , Tunica Media/metabolism , Tunica Media/pathology , Calponins
18.
FASEB J ; 18(11): 1264-6, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15180964

ABSTRACT

Angiogenesis, new capillary blood vessel formation, is essential for embryonic development, wound healing, and cancer growth. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating endothelial cell migration and proliferation. Serum response factor (SRF) is a transcription factor important for embryonic development and activation of immediate early gene expression. The roles of SRF in endothelial cell biology and angiogenesis have not been explored. Here we demonstrate that SRF is a downstream mediator of VEGF signaling in endothelial cells and a critical requirement for VEGF-induced angiogenesis. Knockdown of SRF protein levels in human and rat endothelial cells abolished VEGF-induced in vitro angiogenesis, impaired endothelial cell migration and proliferation, and inhibited VEGF-induced actin polymerization and immediate early gene expression. Injection of SRF antisense expression plasmid into gastric ulcers in rats significantly inhibited in vivo angiogenesis in granulation tissue. Mechanistically, this study also revealed that VEGF promotes SRF expression and nuclear translocation and increases SRF binding activity to DNA in endothelial cells through both Rho-actin and MEK-ERK dependent signaling pathways. These findings have potential therapeutic implications, e.g., local anti-SRF treatment may inhibit angiogenesis crucial for tumor growth.


Subject(s)
Endothelial Cells/physiology , Endothelium, Vascular/cytology , Neovascularization, Physiologic/physiology , Serum Response Factor/physiology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/physiology , Acetic Acid/toxicity , Actins/analysis , Animals , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cells, Cultured/physiology , Collagen , Culture Media, Serum-Free , DNA, Recombinant/administration & dosage , DNA, Recombinant/therapeutic use , Drug Combinations , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Genetic Therapy , Humans , Injections, Intralesional , Laminin , Neovascularization, Physiologic/drug effects , Oligodeoxyribonucleotides, Antisense/pharmacology , Proteoglycans , Rats , Recombinant Proteins/pharmacology , Serum Response Factor/antagonists & inhibitors , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Signal Transduction/physiology , Stomach/blood supply , Stomach Ulcer/chemically induced , Stomach Ulcer/metabolism , Stomach Ulcer/therapy , Umbilical Veins , Vascular Endothelial Growth Factor A/pharmacology
19.
J Neurosci ; 24(9): 2277-85, 2004 Mar 03.
Article in English | MEDLINE | ID: mdl-14999078

ABSTRACT

Recent studies indicate that neuroprotection afforded by brain-derived neurotrophic factor (BDNF) is mediated by extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K). However, the mechanisms by which ERK and PI3K exert neuroprotection are not completely understood. Because ERK1/2 and PI3K both stimulate serum response element (SRE)-mediated gene expression, and serum response factor (SRF) is indispensable for SRE-mediated transcription, we investigated whether SRF contributes to ERK1/2 and PI3K neuroprotection. To accomplish this goal, we used an established experimental paradigm in which BDNF protects postnatal cortical neurons against both trophic deprivation and camptothecin-induced DNA damage. BDNF protection against camptothecin is mediated primarily by ERK1/2 activation, whereas its protection against trophic deprivation is mainly through stimulation of the PI3K pathway (Hetman et al., 1999). Here we demonstrate that expression of a wild-type SRF is sufficient to protect postnatal cortical neurons against camptothecin or trophic deprivation. Expression of a dominant-negative SRF partially reversed BDNF neuroprotection against both apoptotic insults. Moreover, the dominant-negative SRF inhibited neuroprotection against trophic withdrawal afforded by expression of a constitutive active PI3K. In addition, protection against camptothecin by expression of constitutive active mitogen-activated protein kinase kinase 1, an upstream kinase that activates ERK1/2, was also blocked by expression of the dominant-negative SRF. These data suggest that SRF is both necessary and sufficient for BDNF neuroprotection of cortical neurons against trophic deprivation and DNA damage. Our data provide a direct demonstration of a biological function of SRF in neurons and a novel downstream neuroprotective mechanism common to both ERK1/2 and PI3K pathways.


Subject(s)
Neurons/metabolism , Serum Response Factor/physiology , Animals , Brain-Derived Neurotrophic Factor/pharmacology , Camptothecin/antagonists & inhibitors , Camptothecin/pharmacology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , DNA Damage/physiology , Enzyme Activation/drug effects , Gene Expression , Genes, Dominant , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinase Kinases/biosynthesis , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Nerve Growth Factors/pharmacology , Neurons/cytology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Phosphatidylinositol 3-Kinases/drug effects , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, Sprague-Dawley , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Signal Transduction/physiology , Transfection
20.
Am J Respir Cell Mol Biol ; 31(1): 54-61, 2004 Jul.
Article in English | MEDLINE | ID: mdl-14975939

ABSTRACT

Mechanical strain affects airway myocyte phenotype, cytoskeletal architecture, proliferation, and contractile function. We hypothesized that (i) short-term mechanical strain modulates transcription of smooth muscle-specific gene promoters for SM22 and smooth muscle myosin heavy chain (smMHC); and (ii) strain-induced change is mediated by altered actin polymerization in association with activation of protein kinase C (PKC). Primary cultured canine tracheal myocytes were transiently transfected with luciferase reporter plasmids harboring a murine SM22, human smMHC, or artificial serum response factor (SRF)-specific gene promoter and then subjected to cyclic strain for 48 h. This strain protocol significantly reduced transcriptional activity of SM22 and smMHC promoters and an artificial SRF-dependent promoter by 55 +/- 5.9%, 57 +/- 6.4%, and 75 +/- 7.9%, respectively, with concomitant reduction in F/G actin ratio by 31 +/- 8%. PKC inhibitors, GF109203X or Gö6976, significantly attenuated these affects. Similar to strain, strain-independent activation of PKC inhibited SM22, smMHC, and SRF-dependent promoter activity by 61 +/- 4%, 66 +/- 5%, and 28 +/- 15%, respectively, and reduced the F/G actin ratio by 30 +/- 5%. Gel shift assay revealed that PKC activation led to decreased binding of the required transcription factor, SRF, to CArG elements in the SM22 promoter. These data suggest a previously unknown role for PKC isoforms in mechanosensitive signaling in airway myocytes that is associated with coordinated regulation of actin cytoskeletal dynamics and smooth muscle-specific gene transcription.


Subject(s)
Bronchi/enzymology , Gene Expression Regulation, Developmental/genetics , Microfilament Proteins/biosynthesis , Muscle Proteins/biosynthesis , Myocytes, Smooth Muscle/enzymology , Protein Kinase C/metabolism , Actins/biosynthesis , Animals , Bronchi/cytology , Cell Differentiation/genetics , Cells, Cultured , Dogs , Down-Regulation/genetics , Enzyme Inhibitors/pharmacology , Genes, Regulator/genetics , Microfilament Proteins/genetics , Muscle Proteins/genetics , Myosin Heavy Chains/biosynthesis , Myosin Heavy Chains/genetics , Promoter Regions, Genetic/genetics , Respiration/genetics , Serum Response Factor/biosynthesis , Serum Response Factor/genetics , Stress, Mechanical , Transfection
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