Serum Response Factor Expression Research Articles

Targeting serum response factor (SRF) deactivates ΔFosB and mitigates Levodopa-induced dyskinesia in a mouse model of Parkinson's disease.

L-3,4-dihydroxyphenylalanine (L-DOPA) is currently the preferred treatment for Parkinson's Disease (PD) and is considered the gold standard. However, prolonged use of L-DOPA in patients can result in involuntary movements known as Levodopa-induced dyskinesia (LID), which includes uncontrollable dystonia affecting the trunk, limbs, and face. The role of ΔFosB protein, a truncated splice variant of the FosB gene, in LID has been acknowledged, but its underlying mechanism has remained elusive. Here, using a mouse model of Parkinson's disease treated with chronic levodopa we demonstrate that serum response factor (SRF) binds to the FosB promoter, thereby activating FosB expression and levodopa induced-dyskinetic movements. Western blot analysis demonstrates a significant increase in SRF expression in the dyskinetic group compared to the control group. Knocking down SRF significantly reduced abnormal involuntary movements (AIMS) and ΔFosB expression compared to the control. Conversely, overexpression of SRF led to an increase in ΔFosB expression and worsened levodopa-induced dyskinesia. To shed light on the regulatory role of the Akt signaling pathway in this phenomenon, we administered the Akt agonist SC79 to PD mouse models via intraperitoneal injection, followed by L-DOPA administration. The expression of SRF, ΔFosB, and phosphorylated Akt (p-Akt) significantly increased in this group compared to the group receiving normal saline to signify that these happen through Akt signaling pathway. Collectively, our findings identify a promising therapeutic target for addressing levodopa-induced dyskinesia.

Anti-fibrogenic effect of umbilical cord-derived mesenchymal stem cell-conditioned media in human esophageal fibroblasts.

Esophageal fibrosis can develop due to caustic or radiation injuries. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are known to mitigate fibrosis in various organs. However, the potential effects of UC-MSCs on human esophageal fibrosis remain underexplored. This study investigated the anti-fibrogenic properties and mechanisms of UC-MSC-derived conditioned media (UC-MSC-CM) on human esophageal fibroblasts (HEFs). HEFs were treated with TGF-β1 and then cultured with UC-MSC-CM, and the expression levels of extracellular matrix (ECM) components, RhoA, myocardin related transcription factor A (MRTF-A), serum response factor (SRF), Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) were measured. UC-MSC-CM suppressed TGF-β1-induced fibrogenic activation in HEFs, as evidenced by the downregulation of ECM. UC-MSC-CM diminished the expression of RhoA, MRTF-A, and SRF triggered by TGF-β1. In TGF-β1-stimulated HEFs, UC-MSC-CM decreased the nuclear localization of MRTF-A and YAP. Additionally, UC-MSC-CM diminished the TGF-β1-induced nuclear expressions of YAP and TAZ, while concurrently enhancing the cytoplasmic presence of phosphorylated YAP. Furthermore, UC-MSC-CM reduced TGF-β1-induced phosphorylation of Smad2. These findings suggest that UC-MSC-CM may inhibit TGF-β1-induced fibrogenic activation in HEFs by targeting the Rho-mediated MRTF/SRF and YAP/TAZ pathways, as well as the Smad2 pathway. This indicates its potential as a stem cell therapy for esophageal fibrosis.

Serum Response Factor Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

MiR-423-5p Regulates Skeletal Muscle Growth and Development by Negatively Inhibiting Target Gene SRF.

The process of muscle growth directly affects the yield and quality of pork food products. Muscle fibers are created during the embryonic stage, grow following birth, and regenerate during adulthood; these are all considered to be phases of muscle development. A multilevel network of transcriptional, post-transcriptional, and pathway levels controls this process. An integrated toolbox of genetics and genomics as well as the use of genomics techniques has been used in the past to attempt to understand the molecular processes behind skeletal muscle growth and development in pigs under divergent selection processes. A class of endogenous noncoding RNAs have a major regulatory function in myogenesis. But the precise function of miRNA-423-5p in muscle development and the related molecular pathways remain largely unknown. Using target prediction software, initially, the potential target genes of miR-423-5p in the Guangxi Bama miniature pig line were identified using various selection criteria for skeletal muscle growth and development. The serum response factor (SRF) was found to be one of the potential target genes, and the two are negatively correlated, suggesting that there may be targeted interactions. In addition to being strongly expressed in swine skeletal muscle, miR-423-5p was also up-regulated during C2C12 cell development. Furthermore, real-time PCR analysis showed that the overexpression of miR-423-5p significantly reduced the expression of myogenin and the myogenic differentiation antigen (p < 0.05). Moreover, the results of the enzyme-linked immunosorbent assay (ELISA) demonstrated that the overexpression of miR-423-5p led to a significant reduction in SRF expression (p < 0.05). Furthermore, miR-423-5p down-regulated the luciferase activities of report vectors carrying the 3' UTR of porcine SRF, confirming that SRF is a target gene of miR-423-5p. Taken together, miR-423-5p's involvement in skeletal muscle differentiation may be through the regulation of SRF.

Study on attenuation of miR-1-mediated ischemia-induced arrhythmias and infarction in rats by means of fulvning granule

Patients post myocardial infarction (MI) have a high incidence of frequent and complex ventricular arrhythmias. miR-1 is involved in ischemia-induced arrhythmias. Fulvning Granule (FG) is a prescription for treating ischemia-induced arrhythmias. This research investigated therapeutic effect of FG on ischemia-induced arrhythmias in an depth way, focusing on expression of miR-1. 60 healthy Sprague Dawly rats were assigned to operation group, MI+normal saline group, MI+low dose of FG group, MI+moderate dose of FG group and MI+high dose of FG group, MI+propranolol group and MI+moderate dose of FG+propranolol group. Hemodynamic measurement, arrhythmia classification, infarct area evaluation and miR-1 level quantification with expression of PKA and SRF were adopted 4 weeks after operation. FG improved hemodynamic indexes and inhibited expression of miR-1. The optimal dose of FG was medium (P &lt; 0.05). Combination of FG and propranolol further improved the hemodynamics indexes and inhibited the expression of miR-1, PKA and SRF (P &lt; 0.05). FG regulated miR-1 expression via inhibition of Protein Kinase A (PKA) and serum response factor (SRF) expressions. Meanwhile, β-adrenoceptor/PKA signaling pathway played a role in regulating miR-1 expression, while Fulvning granule combined with propranolol and showed an antiarrhythmic role and improved cardiac function after myocardial infarction.

Serum response factor promoting axonal regeneration by activating the Ras-Raf-Cofilin signaling pathway after the spinal cord injury.

Serum response factor (SRF) is important in muscle development, tissue repair, and neuronal regulation. This research aims to thoroughly examine the effects of SRF on spinal cord injury (SCI) and its ability to significantly impact the recovery and regeneration of neuronal axons. The researchers created rat models of SCI and scratch injury to primary spinal cord neurons to observe the expression of relevant factors after neuronal injury. We found that the SRF, Ras, Raf, and cofilin levels increased after injury and gradually returned to normal levels. Afterward, researchers gave rats with SCI an SRF inhibitor (CCG1423) and studied the effects with nuclear magnetic resonance and transmission electron microscopy. The SRF inhibitor rodents had worse spinal cord recovery and axon regrowth than the control group. And the apoptosis of primary neurons after scratch injury was significantly higher in the SRF inhibitor group. Additionally, the researchers utilized lentiviral transfection to modify the SRF expression in neurons. SRF overexpression increased neuron migration while silencing SRF decreased it. Finally, Western blotting and RT-PCR were conducted to examine the expression changes of related factors upon altering SRF expression. The results revealed SRF overexpression increased Ras, Raf, and cofilin expression. Silencing SRF decreased Ras, Raf, and Cofilin expression. Based on our research, the SRF promotes axonal regeneration by activating the "Ras-Raf-Cofilin" signaling pathway.

WAVE2 Is a Vital Regulator in Myogenic Differentiation of Progenitor Cells through the Mechanosensitive MRTFA-SRF Axis.

Skeletal myogenesis is an intricate process involving the differentiation of progenitor cells into myofibers, which is regulated by actin cytoskeletal dynamics and myogenic transcription factors. Although recent studies have demonstrated the pivotal roles of actin-binding proteins (ABPs) as mechanosensors and signal transducers, the biological significance of WAVE2 (Wiskott-Aldrich syndrome protein family member 2), an ABP essential for actin polymerization, in myogenic differentiation of progenitor cells has not been investigated. Our study provides important insights into the regulatory roles played by WAVE2 in the myocardin-related transcription factor A (MRTFA)-serum response factor (SRF) signaling axis and differentiation of myoblasts. We demonstrate that WAVE2 expression is induced during myogenic differentiation and plays a pivotal role in actin cytoskeletal remodeling in C2C12 myoblasts. Knockdown of WAVE2 in C2C12 cells reduced filamentous actin levels, increased globular actin accumulation, and impaired the nuclear translocation of MRTFA. Furthermore, WAVE2 depletion in myoblasts inhibited the expression and transcriptional activity of SRF and suppressed cell proliferation in myoblasts. Consequently, WAVE2 knockdown suppressed myogenic regulatory factors (i.e., MyoD, MyoG, and SMYD1) expressions, thereby hindering the differentiation of myoblasts. Thus, this study suggests that WAVE2 is essential for myogenic differentiation of progenitor cells by modulating the mechanosensitive MRTFA-SRF axis.

The lysine methyltransferase SMYD2 facilitates neointimal hyperplasia by regulating the HDAC3–SRF axis

Coronary restenosis is an important cause of poor long-term prognosis in patients with coronary heart disease. Here, we show that lysine methyltransferase SMYD2 expression in the nucleus is significantly elevated in serum- and PDGF-BB-induced vascular smooth muscle cells (VSMCs), and in tissues of carotid artery injury-induced neointimal hyperplasia. Smyd2 overexpression in VSMCs (Smyd2-vTg) facilitates, but treatment with its specific inhibitor LLY-507 or SMYD2 knockdown significantly inhibits VSMC phenotypic switching and carotid artery injury-induced neointima formation in mice. Transcriptome sequencing revealed that SMYD2 knockdown represses the expression of serum response factor (SRF) target genes and that SRF overexpression largely reverses the inhibitory effect of SMYD2 knockdown on VSMC proliferation. HDAC3 directly interacts with and deacetylates SRF, which enhances SRF transcriptional activity in VSMCs. Moreover, SMYD2 promotes HDAC3 expression via tri-methylation of H3K36 at its promoter. RGFP966, a specific inhibitor of HDAC3, not only counteracts the pro-proliferation effect of SMYD2 overexpression on VSMCs, but also inhibits carotid artery injury-induced neointima formation in mice. HDAC3 partially abolishes the inhibitory effect of SMYD2 knockdown on VSMC proliferation in a deacetylase activity-dependent manner. Our results reveal that the SMYD2-HDAC3-SRF axis constitutes a novel and critical epigenetic mechanism that regulates VSMC phenotypic switching and neointimal hyperplasia.

SAT633 The RNA Helicase UPF1 Mediates AR- And SRF-dependent Transcription In Prostate Cancer

Abstract Disclosure: G. Chauhan: None. V. Venkadakrishnan: None. U. Dahiya: None. Y. Ghanem: None. G. Srinivasan: None. B. Willard: None. Q. Hu: None. E. Cortes: None. S. Liu: None. H. Heemers: None. The ligand-activated androgen receptor (AR) is a transcription factor that drives prostate cancer (CaP). Blocking androgen-activation of AR via androgen deprivation therapy (ADT) is the default treatment for metastatic CaP. Despite initial remission, ADT invariably fails and CaP progresses to castration-recurrent CaP (CRPC), which still relies on aberrantly activated AR. Alternative approaches are needed to inhibit AR action in CaP that has failed ADT. Our lab has been exploring the therapeutic potential of a non-canonical AR signaling mechanism wherein AR stimulates another transcription factor, Serum Response Factor (SRF). AR-SRF action correlates with CaP progression and is enriched in CRPC. Inhibiting SRF-dependent AR action may be an effective treatment strategy following failure of ADT but remains poorly understood. Previously, we determined that the Rho effector Protein Kinase N1 (PKN1) transduces signaling from AR to SRF. This signaling was mediated by PKN1 and SRF interaction at AR-SRF target genes, whose transcription relied on PKN1’s kinase moiety. In Co-IP, ChIP and qRT-PCR assays, kinase-dead PKN1 prevented SRF-PKN1 interaction, recruitment of PKN1 to SRF target genes and AR-SRF target gene expression. Here, we performed biotin-based Turbo-ID-mediated proximity labeling coupled with mass spectrometry (PLA-MS) to elucidate SRF-PKN1 complex function. We isolated 26 significant and mostly novel PKN1 interactors with preferential roles in transcription, RNA binding and DNA repair. The majority was not previously linked to AR or SRF action or CaP progression. PLA-MS studies which we combined with SRF IP, androgen treatment or nuclear localization independently returned several of these PKN1 interactors. A prominent hit was the RNA helicase UPF1, involved in mRNA surveillance and nonsense-mediated mRNA decay (NMD). Co-IP assays confirmed that both SRF and PKN1 interacted with UPF1 and showed wild-type PKN1 increased while kinase-dead PKN1 decreased UPF1-SRF interactions. Moreover, silencing of UPF1 decreased CRPC cell viability, consistent with effects of SRF and PKN1 loss. UPF1 protein sequence contained 3 putative PKN1 consensus phosphorylation motifs, 2 of which were confirmed in in vitro kinase assays. RNA-Seq studies combined with pathway analyses revealed considerable overlap between AR-dependent UPF1- and PKN1-dependent gene signatures and cell functions. Confirming clinical relevance of the UPF1-SRF-PKN1 interactions, we found overlap in gene expression profiles of CaP specimens that showed high PKN1, high SRF and high UPF1 expression, which was enriched in CRPCs and associated with functions in cell proliferation and cell division. Taken together, our work isolated UPF1, which is druggable, as an entirely novel regulator of clinically relevant AR-SRF-PKN1 action in CaP growth, and suggest a previously unrecognized role for NMD in CaP progression. Presentation: Saturday, June 17, 2023

AR activates YAP/TAZ differentially in prostate cancer.

The Hippo signalling pathway is a master regulator of cell growth, proliferation, and cancer. The transcriptional coregulators of the Hippo pathway, YAP and TAZ, are central in various cancers. However, how YAP and TAZ get activated in most types of cancers is not well understood. Here, we show that androgens activate YAP/TAZ via the androgen receptor (AR) in prostate cancer (PCa), and that this activation is differential. AR regulates YAP translation while inducing transcription of the TAZ encoding gene, WWTR1 Furthermore, we show that AR-mediated YAP/TAZ activation is regulated by the RhoA GTPases transcriptional mediator, serum response factor (SRF). Importantly, in prostate cancer patients, SRF expression positively correlates with TAZ and the YAP/TAZ target genes CYR61 and CTGF We demonstrate that YAP/TAZ are not essential for sustaining AR activity, however, targeting YAP/TAZ or SRF sensitize PCa cells to AR inhibition in anchorage-independent growth conditions. Our findings dissect the cellular roles of YAP, TAZ, and SRF in prostate cancer cells. Our data emphasize the interplay between these transcriptional regulators and their roles in prostate tumorigenesis and highlight how these insights might be exploited therapeutically.

#2892 CRITICAL ROLE OF SERUM RESPONSE FACTOR IN PODOCYTE EPITHELIAL–MESENCHYMAL TRANSITION OF DIABETIC NEPHROPATHY

Abstract Background and Aims To investigate the expression and function of serum response factor (SRF) in podocyte epithelial–mesenchymal transition (EMT) of diabetic nephropathy (DN). Method Expression of SRF, pSRF, synaptopodin, P-cadherin, ZO-1, α-SMA, FSP-1, fibronectin and collagen-1 were examined in podocytes or renal cortex following high glucose. SRF was upregulated by SRF plasmids and downregulated by CCG-1423 to investigate how SRF influence podocyte EMT in DN. Streptozocin was used to generate DM in rats. Results ① High glucose induced EMT and SRF upregulation in podocytes (Figure 1). High glucose suppressed synaptopodin and ZO-1 expression, and induced SRF, pSRF collagen-1, FSP-1 and α-SMA expression. SRF transferred from cytoplasm to nuclei obviously and the level of SRF was also increased in podocytes after high glucose treatment. ② SRF overexpression mediated EMT, migration and barrier dysfunction of podocytes (Figure 2). Overexpression of exogenous SRF reduced epithelial synaptopodin expression, and increased collagen-1, FSP-1, α-SMA and Snail expression in podocytes. Transwell chamber migration assay showed that overexpression of SRF significantly upregulated the migration of podocytes across the pores of transwell filters. Albumin filtration assay showed that albumin easily diffused across the monolayer of podocytes transfected with SRF cDNA. ③ Inhibition of SRF preserved phenotypes of podocytes in vitro (Figure 3). CCG-1423 selectively suppressed the expression of pSRF and SRF in podocytes after high glucose stimulation. Besides, simultaneous treatment of podocytes with CCG-1423 also significantly abolished the reduction of synaptopodin expression and the induction of collagen-1, FSP-1, α-SMA and Snail expression. CCG-1423 also reduced the transfer of SRF from cytoplasm to nucleus in podocytes treated with high glucose. ④ The effect of CCG-1423 in diabetic rats (Figure 4–-5). CCG-1423 significantly abrogated the reduction of synaptopodin expression and the induction of SRF, collagen-1, α-SMA and FSP-1 expression in renal cortex tissues. Masson and PAS staining demonstrated that renal glomerular fibrosis was present in DM group, and after 8 weeks treatment with CCG-1423, renal glomerular fibrosis was dramatically ameliorated. In addition, CCG-1423 significantly preserved P-cadherin expression and suppressed α-SMA and FN expression in DN rats. More importantly, CCG-1423 dramatically decreased 24-h urine protein excretion by about 50%, and increased serum albumin, compared with DM group. Conclusion Together, increased SRF activity provokes podocytes EMT and dysfunction in DN. Targeting SRF by small molecule inhibitor may be an attractive therapeutic strategy for DN.

Actin cytoskeleton depolymerization increases matrix metalloproteinase gene expression in breast cancer cells by promoting translocation of cysteine-rich protein 2 to the nucleus.

The actin cytoskeleton plays a critical role in cancer cell invasion and metastasis; however, the coordination of its multiple functions remains unclear. Actin dynamics in the cytoplasm control the formation of invadopodia, which are membrane protrusions that facilitate cancer cell invasion by focusing the secretion of extracellular matrix-degrading enzymes, including matrix metalloproteinases (MMPs). In this study, we investigated the nuclear role of cysteine-rich protein 2 (CRP2), a two LIM domain-containing F-actin-binding protein that we previously identified as a cytoskeletal component of invadopodia, in breast cancer cells. We found that F-actin depolymerization stimulates the translocation of CRP2 into the nucleus, resulting in an increase in the transcript levels of pro-invasive and pro-metastatic genes, including several members of the MMP gene family. We demonstrate that in the nucleus, CRP2 interacts with the transcription factor serum response factor (SRF), which is crucial for the expression of MMP-9 and MMP-13. Our data suggest that CRP2 and SRF cooperate to modulate of MMP expression levels. Furthermore, Kaplan-Meier analysis revealed a significant association between high-level expression of SRF and shorter overall survival and distant metastasis-free survival in breast cancer patients with a high CRP2 expression profile. Our findings suggest a model in which CRP2 mediates the coordination of cytoplasmic and nuclear processes driven by actin dynamics, ultimately resulting in the induction of invasive and metastatic behavior in breast cancer cells.

MRTF-A/SRF signaling suppresses invasion of oral squamous cell carcinoma.

Serum response factor (SRF) and myocardial-associated transcription factor-A (MRTF-A) had different regulatory effects on the tumorigenesis and development in different cancers. However, the role of MRTF-A/SRF in oral squamous cell carcinoma (OSCC) remains to be determined. CCK-8 assay, cell scratch experiment, and transwell invasion assay were conducted to investigate the effects of MRTF-A/SRF on biological behavior of OSCC cells. The expression pattern and prognostic value of MRTF-A/SRF in OSCC were analyzed based on cBioPortal website and TCGA database. Protein-protein interaction network was visualized to identify protein functions. Go and KEGG pathway analyses were performed to investigate related pathways. The effect of MRTF-A/SRF on epithelial-mesenchymal transformation (EMT) of OSCC cells was explored by western blot assay. Overexpression of MRTF-A/SRF inhibited the proliferation, migration, and invasion of OSCC cells in vitro. High expression of SRF was related to better prognosis of OSCC patients on hard palate, alveolar ridge, and oral tongue. Besides, overexpression of MRTF-A/SRF inhibited the EMT of OSCC cells. SRF was closely related to the prognosis of OSCC. High expression of SRF and its co-activator MRTF-A inhibited proliferation, migration, and invasion of OSCC cells in vitro, possibly via EMT suppression.

Serum Response Factor-Regulated IDO1/Kyn-Ahr Pathway Promotes Tumorigenesis of Oral Squamous Cell Carcinoma

Serum response factor (SRF) regulates pro-carcinogenic genes in various cancers, but its role in oral squamous cell carcinoma (OSCC) remains unclear. SRF expression in 70 OSCC samples was detected via immunohistochemistry. Abundant SRF expressed in OSCC tissues was closely associated with tumor metastasis. SRF-overexpressing OSCC cells were constructed to evaluate how SRF affects OSCC cell tumorigenesis and epithelial-to-mesenchymal transition (EMT) in vitro and in vivo. Overexpressed SRF increased OSCC cell migration and invasion in vitro and tumor growth and invasion in vivo. This promoted EMT, characterized by decreased and increased expression of E- and N-cadherin, respectively. Furthermore, an analysis of RNA sequences of transcriptional targets of SRF showed that SRF transactivated the indoleamine 2, 3-dioxygenase 1 (IDO1)/kynurenine-aryl hydrocarbon receptor (Kyn-AhR) signaling pathway in OSCC cell lines. Direct SRF binding to the IDO1 gene promoter upregulated transcription, which was detected through chromatin immunoprecipitation and dual luciferase reporter assays. Inhibiting IDO1 or AhR impaired SRF-induced migration and invasion and prevented EMT in OSCC cells. Our results demonstrated that SRF is a critical regulator of the IDO1/Kyn-AhR signaling pathway. This in turn increases OSCC cell migration and invasion by modulating EMT, which, consequently, favors OSCC cell growth and metastasis. We revealed a novel molecular mechanism through which SRF modulates OSCC metastasis. This should provide potential targets or biomarkers for OSCC diagnosis and treatment.

Serum response factor promotes axon regeneration following spinal cord transection injury.

Studies have shown that serum response factor is beneficial for axonal regeneration of peripheral nerves. However, its role after central nervous system injury remains unclear. In this study, we established a rat model of T9-T10 spinal cord transection injury. We found that the expression of serum response factor in injured spinal cord gray matter neurons gradually increased with time, reached its peak on the 7th day, and then gradually decreased. To investigate the role of serum response factor, we used lentivirus vectors to overexpress and silence serum response factor in spinal cord tissue. We found that overexpression of serum response factor promoted motor function recovery in rats with spinal cord injury. Qualitative observation of biotinylated dextran amine anterograde tracing showed that overexpression of serum response factor increased nerve fibers in the injured spinal cord. Additionally, transmission electron microscopy showed that axon and myelin sheath morphology was restored. Silencing serum response factor had the opposite effects of overexpression. These findings suggest that serum response factor plays a role in the recovery of motor function after spinal cord injury. The underlying mechanism may be related to the regulation of axonal regeneration.

11S Proteasome Activator REGγ Promotes Aortic Dissection by Inhibiting RBM3 (RNA Binding Motif Protein 3) Pathway.

Aortic dissection (AD) is a life-threatening cardiovascular disorder with high mortality and lacking underlying mechanisms or effective treatments. REGγ, the 11S proteasome activator known to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner, emerges as a new regulator in the cardiovascular system. Using β-aminopropionitrile (BAPN)-subjected REGγ knockout AD mice and Ang II (angiotensin II)-treated REGγ deficiency vascular smooth muscle cells (VSMCs) to explore the effect of REGγ in AD progression. REGγ was upregulated in mouse aorta of β-aminopropionitrile-induced AD model in vivo and Ang II-treated VSMCs in vitro. REGγ deficiency ameliorated AD progression in β-aminopropionitrile-induced mice by protecting against the switch in VSMCs from contractile to synthetic phenotype through suppressing RBM3 (RNA-binding motif protein 3) decay. Mechanically, REGγ interacted with and degraded the RNA-binding protein RBM3 directly, leading to decreased mRNA stability, lowered expression and transcriptional activity of transcription factor SRF (serum response factor), subsequently reduced transcription of VSMCs-specific contractile genes, α-SMA (alpha-smooth muscle actin) and SM22α (smooth muscle 22 alpha), caused the switch in VSMCs from contractile to synthetic phenotype and associated AD progression. Ablation of endogenous SRF or RBM3, or overexpressing exogenous RBM3 in VSMCs significantly blocked or reestablished the REGγ-dependent action on VSMCs phenotypic switch of Ang II stimulation in vitro. Furthermore, exogenously introducing RBM3 improved the switch in VSMCs from contractile to synthetic phenotype and associated AD features caused by REGγ in vivo. Our results demonstrated that REGγ promoted the switch in VSMCs from contractile to synthetic phenotype and AD progression by inhibiting RBM3-SRF pathway, indicated that modulating REGγ-proteasome activity may be a potential therapeutic approach for AD-associated cardiovascular dysfunction.

Empagliflozin alleviates diabetic cardiomyopathy via inhibiting excessive autophagy and adverse remodeling in type 1 diabetic mice

Abstract Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with and without diabetes mellitus, but underlying mechanisms remain unclear. Autophagy is an important cardioprotective mechanism under acute stress conditions, but excessive autophagy can accelerate myocardial cell death leading to autosis. In this study we evaluated the role of empagliflozin (EMPA) against cardiac injury in streptozotocin (STZ)-induced diabetic mice, and assessed underlying mechanisms. EMPA (10 mg/kg/die)-treated diabetic male mice had a lower number of TUNEL-positive apoptotic cells (%TdT+ 4.9±2, p&amp;lt;0.05), lower percentage of SA-β-gal positive senescent cardiac area (%SA-β-gal 7.9±1, p&amp;lt;0.05), lower fibrotic area (%picrosirius red 0.15±0.06, p&amp;lt;0.05), lower autophagy (%green fluorescence 2.3±0.6, p&amp;lt;0.01), compared to diabetic mice (%TdT+ 4.9±2; %SA-β-gal 10±2; %picrosirius red fibrotic area 0.24±0.08; %green fluorescence autophagic area 3.98±0.5). Proteomics analysis of cardiac tissue showed downregulation of apoptosis, necrosis and 5' adenosine monophosphate-activated protein kinase (AMPK) pathways, along with upstream activation of vascular endothelial growth factor and sirtuins in the heart of EMPA+STZ treated mice compared to STZ mice. Since activation of sirtuins leads to modulation of cardiomyogenic transcription factors, we analyzed total expression of serum response factor (SRF), myocardin and myocardin-related transcription factor (MRTF), and DNA binding activity of SRF by electromobility shift assay. While EMPA only increased MRTF, EMPA (2.2±0.01, p&amp;lt;0.01) and EMPA+STZ (2.0±0.1, p&amp;lt;0.01) significantly increased SRF/SRE binding activity compared with STZ alone (0.5±0.01), which was paralleled by the increase in cardiac actin expression (4.06±0.1 vs 2.2±0.01, p&amp;lt;0.01). EMPA significantly reversed STZ-induced systolic cardiac dysfunction at echocardiography, and downregulation of pS368-connexin 43 active isoform (Table). In parallel in vitro experiments, EMPA inhibited excessive autophagy in high glucose-treated cardiomyocytes by inhibiting the autophagy inducer GSK3β, leading to reactivation of transcriptional complex for cardiomyocyte contractile genes. Our results indicate that EMPA plays a cardioprotective effect by inhibiting excessive autophagy through the mTOR/AMPK axis in the mouse model of STZ-induced type 1 diabetes. EMPA also promotes cardiac reverse remodeling by inhibiting fibrosis, activates cardiac Cx43 and the promyogenic transcriptional program through the sirtuins-SRF axis (Figure 1), in the context of diabetes. Funding Acknowledgement Type of funding sources: None.

Transforming Growth Factor-β Receptor–Mediated, p38 Mitogen-Activated Protein Kinase–Dependent Signaling Drives Enhanced Myofibroblast Differentiation during Skin Wound Healing in Mice Lacking Hyaluronan Synthases 1 and 3

Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-β receptor-mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-β activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-β activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-β receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-β activation.

Young CSF restores oligodendrogenesis and memory in aged mice via Fgf17.

Recent understanding of how the systemic environment shapes the brain throughout life has led to numerous intervention strategies to slow brain ageing1-3. Cerebrospinal fluid (CSF) makes up the immediate environment of brain cells, providing them with nourishing compounds4,5. We discovered that infusing young CSF directly into aged brains improves memory function. Unbiased transcriptome analysis of the hippocampus identified oligodendrocytes to be most responsive to this rejuvenated CSF environment. We further showed that young CSF boosts oligodendrocyte progenitor cell (OPC) proliferation and differentiation in the aged hippocampus and in primary OPC cultures. Using SLAMseq to metabolically label nascent mRNA, we identified serum response factor (SRF), a transcription factor that drives actin cytoskeleton rearrangement, as a mediator of OPC proliferation following exposure to young CSF. With age, SRF expression decreases in hippocampal OPCs, and the pathway is induced by acute injection with young CSF. We screened for potential SRF activators in CSF and found that fibroblast growth factor 17 (Fgf17) infusion is sufficient to induce OPC proliferation and long-term memory consolidation in aged mice while Fgf17 blockade impairs cognition in young mice. These findings demonstrate the rejuvenating power of young CSF and identify Fgf17 as a key target to restore oligodendrocyte function in the ageing brain.

POS-081 SERUM RESPONSE FACTOR, A NOVEL EARLY DIAGNOSTIC BIOMARKER OF ACUTE KIDNEY INJURY

Studies have shown that serum response factor (SRF) is increased in chronic kidney injury, such as diabetic nephropathy, hyperuricemic nephropathy and renal cell carcinoma. The objective is to explore the early diagnostic value of SRF in acute kidney injury (AKI). AKI-related microarray data were analyzed, and the expression and location of SRF were investigated in the early phase of AKI. ① The microarray analysis results of bilateral I/R mice There were 567 DEGs at 2 h and 857 DEGs at 4 h after I/R in GSE98622 (Figure 1A, 1B). SRF started to increase at 2 h-4 h and decreased to the basal level at 72 h after bilateral I/R in mice (Figure 1C), which suggested that SRF might be connected with AKI. Moreover, as shown in Figure 1C, SRF was increased again 7 d after I/R, which indicated that SRF might be a promising biomarker of the transformation from AKI to chronic kidney injury and post-AKI fibrosis. ② SRF was upregulated and localized in TECs in I/R rats As shown by IHC and IF, SRF was upregulated and located in TECs 6 h after I/R compared to the sham operation group and the control group (Figure 2). This result was consistent with the microarray data from I/R mice. ③ Scr level and renal SRF mRNA expression in I/R rats As shown in Figure 3A, the expression of SRF mRNA in the kidney changed 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h after I/R. In other words, the SRF level in the kidney of the I/R rats started to increase after 3 h, reached its peak at 6-9 h and decreased at 12 h and 24 h. The fitting function estimated that the peak SRF level should be at 6 h. No significant variation in SRF was detected in the sham operation group (Figure 3A). Compared to the increase in SRF levels, the increase in Scr levels was obviously delayed. Scr started to increase after 24 h in the I/R group compared to the control group and the sham group (Figure 3B), indicating that SRF may be an earlier biomarker for AKI and may assist physicians in diagnosing AKI in a timelier manner. ④ The protein level of SRF in kidney, urine and serum in vivo SRF levels in the kidney increased at 1 h, reached its peak at 6 h and started to decline at 12 h after I/R (Figure 4A, 4B). SRF was dramatically increased in the serum of I/R rats at 1 h-6 h after I/R (Figure 4C). In urine, the expression of SRF in the I/R group was dramatically increased at 3 h, reached its peak at 6-9 h, and began to decrease at 12 h after I/R. Additionally, SRF levels were also increased postoperatively in the kidney, urine and serum of the sham rats, but the increases in the sham group were significantly less than those in the I/R group. ⑤ The ROC curve of renal SRF mRNA, renal SRF protein, urinary SRF, serum SRF and Scr in early (before 24 h) AKI To explore more sensitive and specific biomarkers to diagnose AKI, we used a ROC curve assay to investigate the cutoff value of renal SRF mRNA, renal SRF protein, urinary SRF, serum SRF, and Scr levels (Figure 5). The AUCs of SRF mRNA, SRF protein, urinary SRF and serum SRF were 87.9%, 83%, 81.3% and 78.8%, respectively, which were all better than that of Scr (68.8%), which indicated that urinary SRF could be an early diagnostic marker of AKI with better sensitivity and specificity than Scr. SRF is remarkably upregulated in early (before 24 h) AKI and can replace Scr as a potential new early diagnostic biomarker of AKI.