Overexpression Of Serum Response Factor 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.

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.

TRIM28 Regulates Proliferation of Gastric Cancer Cells Partly Through SRF/IDO1 Axis.

Background: Gastric cancer (GC) is one of the most common malignancies worldwide, with high incidence and mortality rate. Tripartite motif-containing 28 (TRIM28) is an important molecule that affects the occurrence and development of tumors, but its function in GC has not been elucidated clearly. The purpose of this study is to explore the molecular mechanism by which TRIM28 affect the GC. Methods: TRIM28 expression was tested in RNA-seq data from TCGA database, tumor tissue samples from patients and GC cell lines. Genes were silenced or overexpressed by siRNA, lentivirus-mediated shRNA, or plasmids. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to explore the proliferation of GC cells after TRIM28 knockdown. RNA-seq and TCGA database were used to identify target genes. Luciferase report assay was employed to detect the possible mechanism between TRIM28 and Indoleamine 2,3-dioxygenase (IDO1). Tryptophan concentration in cell supernatant was measured using a fluorometric assay kit. MGC-803 and 746T cells were injected into mice to establish xenograft animal models. Results: The expression of TRIM28 was positively correlated with tumor size and poorer prognosis. Upregulation of TRIM28 was observed in GC tissues and cells. In vitro, we proved that knockdown of TRIM28 significantly inhibited the proliferation of GC cells. Then TRIM28 was found to be positively correlated with the expression of IDO1 in GC cells. In accordance with this, tryptophan levels in cell supernatants were increased in TRIM28 knockdown GC cells and overexpression of IDO1 could reverse this phenotype. Serum response factor (SRF), a reported regulator of IDO1, was also regulated by TRIM28 in GC cells. And decreased expression of IDO1 induced by TRIM28 knockdown could be partly reversed through overexpression of serum response factor (SRF) in GC cells. Functional research demonstrated that the expression of IDO1 was increased in GC and IDO1 knockdown could also inhibited the proliferation of GC cells. Furthermore, overexpression of IDO1 could partly reverse proliferation inhibited by TRIM28 knockdown in GC cells. In vivo, knockdown of TRIM28 significantly inhibited the tumor growth and overexpression of IDO1 and SRF both could reverse proliferation inhibited by TRIM28 knockdown. Conclusions: TRIM28 is crucial in the development of GC, and may regulate IDO1 through SRF. TRIM28 promote GC cell proliferation through SRF/IDO1 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.

#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.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulatedin calcific areas. We further demonstrated TCF21 expression wasincreased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1β and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

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.

Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem and progenitor cell engraftment

Bone marrow engraftment of the hematopoietic stem and progenitor cells (HSPCs) involves homing to the vasculatures and lodgment to their niches. How HSPCs transmigrate from the vasculature to the niches is unclear. Here, we show that loss of diaphanous-related formin mDia2 leads to impaired engraftment of long-term hematopoietic stem cells and loss of competitive HSPC repopulation. These defects are likely due to the compromised trans-endothelial migration of HSPCs since their homing to the bone marrow vasculatures remained intact. Mechanistically, loss of mDia2 disrupts HSPC polarization and induced cytoplasmic accumulation of MAL, which deregulates the activity of serum response factor (SRF). We further reveal that beta2 integrins are transcriptional targets of SRF. Knockout of beta2 integrins in HSPCs phenocopies mDia2 deficient mice. Overexpression of SRF or beta2 integrins rescues HSPC engraftment defects associated with mDia2 deficiency. Our findings show that mDia2-SRF-beta2 integrin signaling is critical for HSPC lodgment to the niches.

MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1

MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle. Using miRNA sequencing and bioinformatics analysis, we previously found that the miRNA miR-664-5p is significantly differentially expressed in longissimus dorsi muscles of Rongchang pigs. However, the molecular mechanism by which miR-664-5p regulates myogenesis remains unclear. In this study, using flow cytometry, 5-ethynyl-2'-deoxyuridine staining, and cell count and immunofluorescent assays, we found that cell-transfected miR-664-5p mimics greatly promoted proliferation of C2C12 mouse myoblasts by increasing the proportion of cells in the S- and G2-phases and up-regulating the expression of cell cycle genes. Moreover, miR-664-5p inhibited myoblast differentiation by down-regulating myogenic gene expression. In contrast, miR-664-5p inhibitor repressed myoblast proliferation and promoted myoblast differentiation. Mechanistically, using dual-luciferase reporter gene experiments, we demonstrated that miR-664-5p directly targets the 3'-UTR of serum response factor (SRF) and Wnt1 mRNAs. We also observed that miR-664-5p inhibits both mRNA and protein levels of SRF and Wnt1 during myoblast proliferation and myogenic differentiation, respectively. Furthermore, the activating effect of miR-664-5p on myoblast proliferation was attenuated by SRF overexpression, and miR-664-5p repressed myogenic differentiation by diminishing the accumulation of nuclear β-catenin. Of note, miR-664-5p's inhibitory effect on myogenic differentiation was abrogated by treatment with Wnt1 protein, the key activator of the Wnt/β-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/β-catenin signaling pathways in myogenesis.

Overexpression of serum response factor is correlated with poor prognosis in patients with gastric cancer

Serum response factor (SRF) is highly expressed in many tumors, including gastric cancer. However, the exact prognostic utility of SRF in patients with gastric cancer remains unclear. Therefore, in this study, we investigated the expression and prognostic value of SRF in patients with gastric cancer. SRF expression was detected by immunohistochemistry in 149 gastric cancer samples. The relationship between SRF expression and clinicopathological characteristics along with the prognostic significance of SRF in disease-free survival and overall survival of patients was analyzed. We found that the expression of SRF was significantly increased in gastric cancer tissues compared with adjacent noncancerous tissues. Overexpression of SRF was significantly correlated with histologic differentiation, invasion depth, lymph node metastasis, and TNM stage. Furthermore, disease-free survival rate and overall survival rate were both significantly lower for patients with high SRF expression. Multivariate Cox regression analysis identified high SRF expression as an independent prognostic factor for disease-free survival but not for overall survival. Our findings indicate that overexpression of SRF may play an important role in human gastric cancer recurrence and prognosis. SRF may serve as a predictive marker for prognosis of gastric cancer.

Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor

After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal’s lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.

Serum response factor induces endothelial-mesenchymal transition in glomerular endothelial cells to aggravate proteinuria in diabetic nephropathy.

We investigated the expression and function of serum response factor (SRF) in endothelial-mesenchymal transition (EndMT) in glomerular endothelial cells (GEnCs) of diabetic nephropathy (DN). The expression of SRF, endothelial markers (VE-cadherin, CD31), and mesenchymal markers (α-SMA, FSP-1, fibronectin) was examined in GEnCs following high glucose or in renal cortex tissues of DN rats. SRF was upregulated by SRF plasmids and downregulated by CCG-1423 (a small molecule inhibitor of SRF) to investigate how SRF influenced EndMT in GEnCs of DN. Streptozocin (STZ) was used to generate diabetes mellitus DM in rats. In GEnCs after high glucose treatment and in renal cortex tissues of diabetic rats, SRF, α-SMA, FSP-1, and fibronectin increased, while VE-cadherin and CD31 declined. SRF overexpression in GEnCs induced expression of Snail, an important transcription factor mediating EndMT. Blockade of SRF reduced Snail induction, protected GEnCs from EndMT, and ameliorated proteinuria. Together, increased SRF activity provokes EndMT and barrier dysfunction of GEnCs in DN. Targeting SRF by small molecule inhibitor may be an attractive therapeutic strategy for DN.

Myocardin-related transcription factor A (MRTFA) regulates the fate of bone marrow mesenchymal stem cells and its absence in mice leads to osteopenia

ObjectiveArising from common progenitors in the bone marrow, adipogenesis and osteogenesis are closely associated yet mutually exclusive during bone marrow mesenchymal stem cell (BMSC) development. Previous studies have shown that morphological changes can affect the early commitment of pluripotent BMSCs to the adipose versus osteoblastic lineage via modulation of RhoA activity. The RhoA pathway regulates actin polymerization to promote the incorporation of globular actin (G-actin) into filamentous actin (F-actin). In doing so, myocardin-related transcription factors (MRTFs) dissociate from bound G-actin and enter the nucleus to co-activate serum response factor (SRF) target gene expression. In this study, we investigated whether MRTFA/SRF is acting downstream of the RhoA pathway to regulate BMSC commitment in mice. MethodsThe effects of knocking out MRTFA on skeletal homeostasis was studied in MRTFA KO mice using micro-CT, QPCR and western blot assays. To determine how MRTFA affects the mechanisms regulating BMSC fate decisions, primary bone marrow stromal cells from WT and MRTFA KO mice as well as C3H10T1/2 cell lines were analyzed in vitro. ResultsGlobal MRTFA KO mice have lower whole body weight, shorter femoral and tibial lengths as well as significantly decreased bone mass in their femurs. BMSCs isolated from the KO mice show increased adipogenesis and reduced osteogenesis when compared to WT littermates. KO mice, particularly females, develop osteopenia with age, and this was enhanced by a high fat diet. Over-expression of MRTFA or SRF enhances osteogenesis in CH310T1/2 cell lines. Sca1+, CD45− cells from KO marrow express lower amounts of smooth muscle actin (SMA) and TAZ/YAP target genes compared to WT counterparts. ConclusionThis study identified MRTFA as a novel regulator of skeletal homeostasis by regulating the balance between adipogenic and osteogenic differentiation of BMSCs. We propose that MRTFA promotes the osteogenic activity of TAZ/YAP by maintaining SMA production in BMSCs.

SRF promotes gastric cancer metastasis through stromal fibroblasts in an SDF1-CXCR4-dependent manner.

It has been suggested that the overexpression of serum response factor (SRF) in cancer cells may promote cancer metastasis. However, the exact pathway by which SRF promotes metastasis has not been clarified. Here we showed that SRF promotes gastric cancer (GC) metastasis through stromal fibroblasts in an SDF1-CXCR4-dependent manner. SRF expression was significantly increased in metastatic GCs compared with the non-metastatic GCs (n=50, p=0.013). Immuno-staining indicated that SRF was primarily expressed in a-smooth muscle actin (αSMA)-expressing periglandular fibroblasts in GCs. The conditioned medium (CM) from CCD18Co fibroblasts stably transfected with the SRF vector (CCD18Co-SRF) significantly enhanced migration of MKN45 gastric cancer cells. In contrast, the CM from CCD18Co fibroblasts, in which SRF was downregulated, inhibited mobility of MKN45 cells. Similar results were observed in cultured BGC823 cells even when they were treated with the NIH3T3-SRF CM. When MKN45 cells and SRF-upregulated or downregulated CCD18Co cells were simultaneously co-injected into the tail veins of NOD-SCID mice, a significant increase or decrease was, respectively, observed in the experimental pulmonary metastasis of cancer cells. Furthermore, SRF overexpression significantly upregulated `SMA and stromal cell derived factor1 (SDF1) expression in these fibroblasts, and an anti-SDF1 antibody or the SDF1 receptor CXCR4-specific inhibitor AMD3100 treatment completely reversed the SRF-enhanced migration of cancer cells. Quantitative RT-PCR demonstrated that the expression level of SRF was positively correlated with that of SDF1 in 92 GC samples (r=0.63, p<0.001). In conclusion, SRF promote GC metastasis by facilitating myofibroblast-cancer cell crosstalk in an SDF1-CXCR4 dependent manner, and maybe a therapeutic target.

Serum response factor provokes epithelial-mesenchymal transition in renal tubular epithelial cells of diabetic nephropathy.

We investigated the role of serum response factor (SRF) in epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs) in diabetic nephropathy (DN). The expression of SRF, epithelial markers (E-cadherin and ZO-1), and mesenchymal markers (fibronectin, collagen-1, α-SMA, FSP-1) was examined in human proximal renal tubular epithelial cells (HK-2 cells) or renal medulla tissues following high glucose. SRF was upregulated by SRF plasmids and downregulated by CCG-1423 (a small molecule inhibitor of SRF) to investigate how SRF influenced EMT in TECs of DN. Streptozotocin was used to generate DM in rats. In HK-2 cells after high-glucose treatment and renal medulla tissues of diabetic rats, SRF, fibronectin, collagen-1, α-SMA, and FSP-1 increased, while E-cadherin and ZO-1 declined. SRF overexpression in HK-2 cells induced expression of Snail, an important transcription factor mediating EMT. Blockade of SRF by CCG-1423 reduced Snail induction and protected TECs from EMT both in vitro and in vivo. Together, increased SRF activity promotes EMT in TECs and dysfunction in DN. Targeting SRF by small molecule inhibitor may be an attractive therapeutic strategy for DN.

Serum Response Factor Protects Retinal Ganglion Cells Against High-Glucose Damage.

Serum response factor (SRF), which encodes the MADS-box family of related proteins, is a common transcription factor related to the expression of genes associated with cell survival. However, SRF's role in retinal ganglion cells (RGCs) after high-glucose injury remains unclear. In this study, we investigate the protective role of SRF after high-glucose injury and its underlying mechanism. The in vitro RGC model subjected to high glucose was established by employing a 50mmol/L glucose culture environment. As detected by real-time quantitative PCR and Western blot, SRF was significantly upregulated in RGCs treated with high glucose. Overexpression of SRF significantly promoted survival among RGCs exposed to high glucose and inhibited RGC apoptosis. Knockdown of SRF exerted an inverse effect. Moreover, SRF upregulation enhanced expression of an antioxidant protein, nuclear factor erythroid 2-related factor (Nrf2), via control of the Fos-related antigen 1 (Fra-1). SRF upregulation also affected RGC survival after high-glucose treatment. Our findings showed that overexpression of SRF promoted survival of RGCs after high-glucose injury by regulating Fra-1 and Nrf2.

Critical role of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy.

To investigate the expression and function of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy. The expression of serum response factor, epithelial markers and mesenchymal markers was examined in podocytes or renal cortex tissues following high glucose. Serum response factor was upregulated by its plasmids and downregulated by CCG-1423 to investigate how it influenced podocyte epithelial-mesenchymal transition in diabetic nephropathy. Streptozotocin was used to generate diabetes mellitus in rats. In podocytes after high glucose treatment, serum response factor and mesenchymal markers increased, while epithelial markers declined. Similar changes were observed in vivo. Serum response factor overexpression in podocytes induced expression of Snail, an important transcription factor mediating epithelial-mesenchymal transition. Blockade of serum response factor reduced Snail induction, protected podocytes from epithelial-mesenchymal transition and ameliorated proteinuria. Together, increased serum response factor activity provokes podocytes' epithelial-mesenchymal transition and dysfunction in diabetic nephropathy. Targeting serum response factor by small-molecule inhibitor may be an attractive therapeutic strategy for diabetic nephropathy.

Overexpression of Serum Response Factor in Neurons Restores Ocular Dominance Plasticity in a Model of Fetal Alcohol Spectrum Disorders.

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. Ferrets received 3.5g/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 5days after which single-unit recordings were conducted to determine whether changes in ocular dominance had occurred. 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. 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.

Serum response factor accelerates the high glucose-induced Epithelial-to-Mesenchymal Transition (EMT) via snail signaling in human peritoneal mesothelial cells.

BackgroundEpithelial-to-Mesenchymal Transition (EMT) induced by glucose in human peritoneal mesothelial cells (HPMCs) is a major cause of peritoneal membrane (PM) fibrosis and dysfunction.MethodsTo investigate serum response factor (SRF) impacts on EMT-derived fibrosis in PM, we isolated HPMCs from the effluents of patients with end-stage renal disease (ESRD) to analyze alterations during peritoneal dialysis (PD) and observe the response of PM to SRF in a rat model.ResultsOur results demonstrated the activation and translocation of SRF into the nuclei of HPMCs under extensive periods of PD. Accordingly, HPMCs lost their epithelial morphology with a decrease in E-cadherin expression and an increase in α-smooth muscle actin (α-SMA) expression, implying a transition in phenotype. PD with 4.25% glucose solution significantly induced SRF up-regulation and increased peritoneal thickness. In immortal HPMCs, high glucose (HG, 60 mmol/L) stimulated SRF overexpression in transformed fibroblastic HPMCs. SRF-siRNA preserved HPMC morphology, while transfection of SRF plasmid into HPMCs caused the opposite effects. Evidence from electrophoretic mobility shift, chromatin immunoprecipitation and reporter assays further supported that SRF transcriptionally regulated Snail, a potent inducer of EMT, by directly binding to its promoter.ConclusionsOur data suggested that activation of SRF/Snail pathway might contribute to the progressive PM fibrosis during PD.

SRF expedites metastasis and modulates the epithelial to mesenchymal transition by regulating miR-199a-5p expression in human gastric cancer.

Dysregulation of transcription factors (TFs) is associated with tumor progression, but little is known about TF expression patterns in the context of gastric cancer (GC) metastasis. Using array-based profile analysis, we found that 22 TFs showed differential activities between GC cell lines with low- and high-metastatic potential. Of this group of TFs, serum response factor (SRF) was significantly upregulated in metastatic GC cells. SRF expression was frequently elevated in a panel of metastatic GC cells and tissues, and high-level expression of SRF was significantly associated with a more aggressive phenotype and poor prognosis in patients with GC. In GC cell lines, overexpression of SRF potently promoted cell migration and invasion in vitro as well as the formation of intrahepatic and pulmonary metastases in vivo, whereas loss of SRF inhibited GC cell invasion and metastasis. We also performed a microRNA microarray to screen for transcriptional targets of SRF and found that SRF transactivates miR-199a-5p and miR-199a-3p by directly binding to their promoters. We further determined that overexpression of miR-199a-5p, but not miR-199a-3p, increased GC cell invasion and metastasis. In contrast, inhibition of miR-199a-5p impaired the metastatic potential of GC cells in vitro and in vivo, and E-cadherin was identified as a direct and functional target of miR-199a-5p in GC cells. Specifically, our results showed that SRF promotes GC metastasis and the epithelial to mesenchymal transition (EMT) though miR-199a-5p-mediated downregulation of E-cadherin. The present study thus provides insight into the specific biological behavior of SRF in GC metastasis. As increased activity of the SRF/miR-199a-5p/E-cadherin pathway appears to promote GC cell EMT and metastasis, these regulators may therefore be developed as therapeutic targets or biomarkers for GC progression.