Dystrophin involvement in peripheral circadian SRF signalling

Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

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Dystrophin regulates peripheral circadian SRF signalling

10.1101/2021.06.23.449492 ◽

2021 ◽

Author(s):

Corinne A Betts ◽

Aarti Jagannath ◽

Tirsa van Westering ◽

Melissa Bowerman ◽

Subhashis Banerjee ◽

...

Keyword(s):

Target Genes ◽

Serum Response Factor ◽

Actin Binding ◽

Peripheral Tissues ◽

Downstream Target ◽

Actin Binding Domain ◽

Muscle Wasting Disease ◽

Dystrophin Protein ◽

First Time ◽

Serum Response

Dystrophin is a sarcolemmal protein essential for muscle contraction and maintenance, absence of which leads to the devastating muscle wasting disease Duchenne muscular dystrophy (DMD). Dystrophin has an actin-binding domain, which specifically binds and stabilises filamentous (F)-actin, an integral component of the RhoA-actin-serum response factor (SRF)-pathway. The RhoA-actin-SRF-pathway plays an essential role in circadian signalling whereby the hypothalamic suprachiasmatic nucleus, transmits systemic cues to peripheral tissues, activating SRF and transcription of clock target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised that dystrophin loss causes circadian deficits. Here we show for the first time alterations in the RhoA-actin-SRF-signalling-pathway, in both dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios and nuclear MRTF, dysregulation of core clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from DMD patients harbouring an array of mutations. Further, disrupted circadian locomotor behaviour was observed in dystrophic mice indicative of disrupted SCN signalling, and indeed dystrophin protein was absent in the SCN of dystrophic animals. Dystrophin is thus a critically important component of the RhoA-actin-SRF-pathway and a novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

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Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion

Nature Communications ◽

10.1038/s41467-021-26839-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sergi Marco ◽

Matthew Neilson ◽

Madeleine Moore ◽

Arantxa Perez-Garcia ◽

Holly Hall ◽

...

Keyword(s):

Nuclear Import ◽

Tyrosine Kinases ◽

Target Genes ◽

Serum Response Factor ◽

Actin Binding ◽

Cell Scattering ◽

Nuclear Localisation Sequence ◽

Related Transcription Factor ◽

Endosomal System ◽

Serum Response

AbstractSignals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that hepatocyte growth factor, an activator of MET (an RTK), promotes Rab17- and clathrin-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2’s cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

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Abstract 2178: Sonic Hedgehog In Adult Hypoxic Cerebellum

Circulation ◽

10.1161/circ.116.suppl_16.ii_472-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Giuseppe Straface ◽

Andrea Flex ◽

Federico Biscetti ◽

Eleonora Gaetani ◽

Giovanni Pecorini ◽

...

Keyword(s):

Sonic Hedgehog ◽

Target Genes ◽

Positive Staining ◽

Lacz Gene ◽

Downstream Target ◽

Shh Pathway ◽

And Migration ◽

First Time ◽

Proliferation And Migration

Background: Cerebellar hypoxia is responsible for important aspects of cognitive deterioration and motor disturbances in neurological disorders, such as stroke, vascular dementia, and neurodegeneration. In the cerebellum, VEGF is significantly upregulated after hypoxia and is able to induce angiogenesis, reduce neuronal apoptosis, and regulate neuronal differentiation, proliferation, and migration. But, VEGF is not sufficient to provide neuroprotection. A crucial role is played by growth associated protein-43 (GAP43), for which important activities have been described. The purpose of this study was to investigate the role of the developmental Sonic hedgehog (Shh) signaling pathway in postnatal hypoxic cerebellum and its relationship with VEGF and GAP43 expression. Methods: We used adult C57BL/6J mice, ptc1-lacZ mice, and GAP43−/− mice for these experiments. Ptc1-lacZ mice carry a non-disruptive insertion of the lacZ gene under the control of the ptc1 promoter. Ptc1 is a downstream-transcriptional target of Shh and its upregulation indicates activation of the Shh pathway. Mice were exposed to systemic normobaric hypoxia (6%O 2 ) for 6 hours and the expression of Shh, Ptc1, VEGF, and GAP43 were investigated. Results: After exposure to hypoxia, Shh-positive staining was detected in Purkinje cells (PCs). The same cells were also lacZ(ptc1)-positive, indicating that PCs are both Shh-producing and -responding elements. Also the cells of the internal granular layer (IGL) were lacZ(ptc1)-positive, indicating that these cells are Shh-responsive. LacZ(ptc1)-positive IGL cells were also immunopositive for VEGF and GAP-43. We also found that ptc1 expression is lost in PCs of GAP43−/− mice, indicating that Shh requires GAP43 to activate its downstream target genes in PCs. Finally, when cultures enriched in granular cells were stimulated with Shh recombinant protein, GAP43 phosphorylation was increased. This effect was inhibited by Shh-inhibitor cyclopamine. Conclusions: This is the first time that hypoxia is reported to activate the Shh pathway in the adult. Our data suggest that the Shh pathway might be important for the cerebellar response to hypoxia, through interactions with VEGF and GAP43.

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Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

Molecular and Cellular Biology ◽

10.1128/mcb.01097-15 ◽

2016 ◽

Vol 36 (10) ◽

pp. 1526-1539 ◽

Cited By ~ 9

Author(s):

Julia Weissbach ◽

Franziska Schikora ◽

Anja Weber ◽

Michael Kessels ◽

Guido Posern

Keyword(s):

Serum Response Factor ◽

De Novo ◽

Actin Dynamics ◽

Actin Binding ◽

Serum Stimulation ◽

Competitive Mechanism ◽

Related Transcription Factor ◽

Simultaneous Inhibition ◽

Serum Response

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actinin vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, andde novoformation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation.

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RAGE‐DIAPH1 modulates hyperglycemia driven induction of Serum response factor target genes in cardiac cells

The FASEB Journal ◽

10.1096/fasebj.2019.33.1_supplement.830.9 ◽

2019 ◽

Vol 33 (S1) ◽

Author(s):

Gopalkrishna Sreejit ◽

Nosirudeen Quadri ◽

Radha Ananthakrishnan ◽

Ann Marie Schmidt ◽

Ravichandran Ramasamy

Keyword(s):

Target Genes ◽

Serum Response Factor ◽

Cardiac Cells ◽

Response Factor ◽

Serum Response

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Nuclear-capture of endosomes drives depletion of nuclear G-actin to promote SRF/MRTF gene expression and cancer cell invasiveness

10.21203/rs.3.rs-50672/v1 ◽

2020 ◽

Author(s):

Sergi Marco ◽

Matthew Neilson ◽

Madeleine Moore ◽

Arantxa Pérez-García ◽

Holly Hall ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Import ◽

Tyrosine Kinases ◽

Target Genes ◽

Serum Response Factor ◽

Actin Binding ◽

Cell Scattering ◽

Nuclear Localisation Sequence ◽

Related Transcription Factor ◽

Cell Invasiveness

Abstract Signals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that an RTK, cMET promotes Rab17-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2’s cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

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Lamina-associated polypeptide 2α is required for intranuclear MRTF-A activity

10.1101/2021.01.08.425886 ◽

2021 ◽

Author(s):

Ekaterina Sidorenko ◽

Maria Sokolova ◽

Antti Pennanen ◽

Salla Kyheroinen ◽

Guido Posern ◽

...

Keyword(s):

Target Genes ◽

Serum Response Factor ◽

Actin Dynamics ◽

Chromatin Binding ◽

Binding Partner ◽

Related Transcription Factor ◽

Direct Binding ◽

Efficient Expression ◽

Serum Response

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for binding of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

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Soluble Epoxide Hydrolase Inhibitors Regulate Ischemic Arrhythmia by Targeting MicroRNA-1

Frontiers in Physiology ◽

10.3389/fphys.2021.717119 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yanying Chen ◽

Qiong Liu ◽

Tian Yang ◽

Li Shen ◽

Danyan Xu

Keyword(s):

Epoxide Hydrolase ◽

Target Genes ◽

Serum Response Factor ◽

Soluble Epoxide Hydrolase ◽

Soluble Epoxide Hydrolase Inhibitors ◽

Interfering Rna ◽

Serum Response ◽

Ischemic Arrhythmia ◽

Antiarrhythmic Effects

Background: Soluble epoxide hydrolase inhibitors (sEHis) inhibit the degradation of epoxyeicosatrienoic acids (EETs) in cells, and EETs have antiarrhythmic effects. Our previous experiments confirmed that t-AUCB, a preparation of sEHis, inhibited ischemic arrhythmia by negatively regulating microRNA-1 (miR-1), but its specific mechanism remained unclear.Aim: This study aimed to examine the role of serum response factor (SRF) and the PI3K/Akt/GSK3β pathway in t-AUCB-mediated regulation of miR-1 and the interaction between them.Methods/Results: We used SRF small interfering RNA (siSRF), SRF small hairpin (shSRF) RNA sequence adenovirus, PI3K/Akt/GSK3β pathway inhibitors, t-AUCB, and 14,15-EEZE (a preparation of EETs antagonists) to treat mouse cardiomyocytes overexpressing miR-1 and mice with myocardial infarction (MI). We found that silencing SRF attenuated the effects on miR-1 and its target genes KCNJ2 and GJA1 in the presence of t-AUCB, and inhibition of the PI3K/Akt/GSK3β pathway antagonized the effects of t-AUCB on miR-1, KCNJ2, and GJA1, which were associated with PI3Kα, Akt, and Gsk3β but not PI3Kβ or PI3Kγ. Moreover, the PI3K/Akt/GSK3β pathway was involved in the regulation of SRF by t-AUCB, and silencing SRF inhibited the t-AUCB-induced increases in Akt and Gsk3β phosphorylation.Conclusions: Both the SRF and the PI3K/Akt/GSK3β pathway are involved in the t-AUCB-mediated regulation of miR-1, and these factors interact with each other.

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Arp5 is a key regulator of myocardin in smooth muscle cells

The Journal of Cell Biology ◽

10.1083/jcb.201307158 ◽

2014 ◽

Vol 204 (5) ◽

pp. 683-696 ◽

Cited By ~ 10

Author(s):

Tsuyoshi Morita ◽

Ken’ichiro Hayashi

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Messenger Rna ◽

Serum Response Factor ◽

Muscle Cells ◽

Actin Binding ◽

Promoter Regions ◽

Muscle Genes ◽

Well Differentiated ◽

Serum Response

Myocardin (Myocd) and Myocd-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). RPEL motifs are monomeric globular actin (G-actin) binding elements that regulate MRTF localization and activity. However, the function of the RPEL motif in Myocd is largely unknown because of its low affinity for G-actin. Here, we demonstrated that the Myocd RPEL motif bound to actin-related protein 5 (Arp5) instead of conventional actin, resulting in a significant suppression of Myocd activity. In addition, Arp5 bound to a DNA binding domain of SRF via its C-terminal sequence and prevented the association of the Myocd–SRF complex with the promoter regions of smooth muscle genes. Well-differentiated smooth muscle cells mainly expressed a specific splicing variant of arp5; therefore, the protein level of Arp5 was markedly reduced by partial messenger RNA decay and translational suppression. In dedifferentiated smooth muscle cells, Arp5 knockdown restored the differentiated phenotype via Myocd activation. Thus, Arp5 is a key regulator of Myocd activity.

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