Serum Response Factor Research Articles

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.

Abstract 454: Validation of GPR35 as a novel cancer target in digestive tract cancers and discovery of potent, selective GPR35 inverse agonists

Abstract Cancers of the digestive tract are a major area of unmet clinical need with incidence rates of some cancers increasing and early onset forms becoming more prevalent. Environmental factors, such as poor diet and microbiota, as well as inflammatory conditions, are strongly associated with occurrence of digestive system cancers. Obesogenic diet combined with microbiotic metabolism can lead to high levels of secondary bile acids in the human gut. Secondary bile acids deoxy- and litho-cholic acid have been shown to have oncogenic potential and their levels are increased in the intestines of cancer patients relative to healthy controls. GPR35 is an orphan class A G protein-coupled receptor primarily expressed in digestive system epithelial tissues and cells of myeloid lineage. GPR35 polymorphisms are strongly associated with inflammatory bowel diseases. In particular, rs37947171, a missense variant that codes for a threonine to methionine substitution at position 108 (T108M) and has been shown to have hypermorphic function, is strongly associated with Crohn’s disease and ulcerative colitis. GPR35 is also overexpressed in adenocarcinomas of the digestive tract, including those of esophageal, hepatic/bile ductal, pancreatic, gastric and colorectal origin, and high expression has been shown to confer poor prognosis in various digestive system cancers. Consistent with a proposed role in digestive tract cancers we now show that GPR35 is activated by lithocholic acid. Furthermore, we demonstrate through CRISPR-gene editing of cancer cells and RNAseq analysis that GPR35 can regulate transcriptional and cytoskeletal modules associated with hallmarks of cancer, including chemokine and growth factor expression and F-actin formation. GPR35 activates a transcriptional program that is enriched for genes containing pro-oncogenic serum response factor response element in their upstream promoters. High throughput screening and medicinal chemistry optimization has led to the discovery of potent antagonists of GPR35 signalling. Pharmacological characterisation of lead series has shown that they are able to prevent Gα and β-arrestin protein binding, while also inhibiting phospho-ERK, calcium flux, receptor internalisation and serum response factor-induced gene transcription. Furthermore, the series are competitive with predicted orthosteric agonist, can block activation of receptor signalling by lithocholic acid and act as inverse agonists of constitutive receptor tone. Profiling these inhibitors in cancer models is currently ongoing. This work provides the basis for the pre-clinical development of GPR35 inverse agonists as anti-cancer drugs. Citation Format: James Westcott, Christopher A. Luckhurst, Grahame McKenzie, Danish Memon, Li-Chiung Lin, Sinead Knight, Elizabeth J. Blaikley, Martin Pearce, Hannah R. Warren, Eleanor Parker, Graeme Milligan, Stuart W. Hughes, Tom McCarthy. Validation of GPR35 as a novel cancer target in digestive tract cancers and discovery of potent, selective GPR35 inverse agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 454.

SRF and SRF Cofactor mRNA Expression Is Differentially Regulated by BDNF Stimulation in Cortical Neurons.

Serum response factor (SRF) is a transcription factor that plays essential roles in multiple brain functions in concert with SRF cofactors such as ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which comprises MKL1/MRTFA and MKL2/MRTFB. Here, we stimulated primary cultured rat cortical neurons with brain-derived neurotrophic factor (BDNF) and investigated the levels of SRF and SRF cofactor mRNA expression. We found that SRF mRNA was transiently induced by BDNF, whereas the levels of SRF cofactors were differentially regulated: mRNA expression of Elk1, a TCF family member, and MKL1/MRTFA were unchanged, while in contrast, mRNA expression of MKL2/MRTFB was transiently decreased. Inhibitor experiments revealed that BDNF-mediated alteration in mRNA levels detected in this study was mainly due to the extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Collectively, BDNF mediates the reciprocal regulation of SRF and MKL2/MRTFB at the mRNA expression level through ERK/MAPK, which may fine-tune the transcription of SRF target genes in cortical neurons. Accumulating evidence regarding the alteration of SRF and SRF cofactor levels detected in several neurological disorders suggests that the findings of this study might also provide novel insights into valuable therapeutic strategies for the treatment of brain diseases.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

ObjectiveVascular smooth muscle cell (VSMC) differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension, atherosclerosis, and restenosis. MicroRNA-146a (miR-146a) has been proven to be involved in cell proliferation, migration, and tumor metabolism. However, little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells (ESCs). This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.MethodsMouse ESCs were differentiated into VSMCs, and the cell extracts were analyzed by Western blotting and RT-qPCR. In addition, luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed. Finally, C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs, and immunohistochemistry, Western blotting, and RT-qPCR assays were carried out on tissue samples from these mice.ResultsmiR-146a was significantly upregulated during VSMC differentiation, accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin (SMαA), smooth muscle 22 (SM22), smooth muscle myosin heavy chain (SMMHC), and h1-calponin. Furthermore, overexpression of miR-146a enhanced the differentiation process in vitro and in vivo. Concurrently, the expression of Kruppel-like factor 4 (KLF4), predicted as one of the top targets of miR-146a, was sharply decreased in miR-146a-overexpressing ESCs. Importantly, inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs. In addition, miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors, including serum response factor (SRF) and myocyte enhancer factor 2c (MEF-2c).ConclusionOur data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

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.

Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH).Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression.Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

A Kinase Interacting Protein 1 (AKIP1) promotes cardiomyocyte elongation and physiological cardiac remodelling

A Kinase Interacting Protein 1 (AKIP1) is a signalling adaptor that promotes physiological hypertrophy in vitro. The purpose of this study is to determine if AKIP1 promotes physiological cardiomyocyte hypertrophy in vivo. Therefore, adult male mice with cardiomyocyte-specific overexpression of AKIP1 (AKIP1-TG) and wild type (WT) littermates were caged individually for four weeks in the presence or absence of a running wheel. Exercise performance, heart weight to tibia length (HW/TL), MRI, histology, and left ventricular (LV) molecular markers were evaluated. While exercise parameters were comparable between genotypes, exercise-induced cardiac hypertrophy was augmented in AKIP1-TG vs. WT mice as evidenced by an increase in HW/TL by weighing scale and in LV mass on MRI. AKIP1-induced hypertrophy was predominantly determined by an increase in cardiomyocyte length, which was associated with reductions in p90 ribosomal S6 kinase 3 (RSK3), increments of phosphatase 2A catalytic subunit (PP2Ac) and dephosphorylation of serum response factor (SRF). With electron microscopy, we detected clusters of AKIP1 protein in the cardiomyocyte nucleus, which can potentially influence signalosome formation and predispose a switch in transcription upon exercise. Mechanistically, AKIP1 promoted exercise-induced activation of protein kinase B (Akt), downregulation of CCAAT Enhancer Binding Protein Beta (C/EBPβ) and de-repression of Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4 (CITED4). Concludingly, we identified AKIP1 as a novel regulator of cardiomyocyte elongation and physiological cardiac remodelling with activation of the RSK3-PP2Ac-SRF and Akt-C/EBPβ-CITED4 pathway. These findings suggest that AKIP1 may serve as a nodal point for physiological reprogramming of cardiac remodelling.

Zinc finger transcription factor Egf1 promotes non-alcoholic fatty liver disease

Background & AimsNon-alcoholic fatty liver disease (NAFLD) contributes to the global epidemic of metabolic syndrome and is considered a prelude to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. During NAFLD pathogenesis, hepatic parenchymal cells (hepatocytes) undergo both morphological and functional changes owing to a rewired transcriptome. The underlying mechanism is not entirely clear. In the present study, we investigated the involvement of early growth response 1 (Egr1) in NAFLD. MethodsQuantitative PCR, Western blotting, and histochemical staining were used to assess gene expression levels. Chromatin immunoprecipitation was used to evaluate protein binding to DNA. NAFLD was evaluated in leptin receptor-deficient (db/db) mice. ResultsWe report here that Egr1 was upregulated by pro-NAFLD stimuli in vitro and in vivo. Further analysis revealed that serum response factor (SRF) was recruited to the Egr1 promoter and mediated Egr1 transactivation. Importantly, Egr1 depletion markedly mitigated NAFLD in db/db mice. RNA sequencing revealed that Egr1 knockdown in hepatocytes, on the one hand, boosted fatty acid oxidation (FAO) and, on the other hand, suppressed the synthesis of chemoattractants. Mechanistically, Egr1 interacted with peroxisome proliferator-activated receptor α (PPARα) to repress PPARα-dependent transcription of FAO genes by recruiting its co-repressor NGFI-A binding protein 1 (Nab1), which potentially led to promoter deacetylation of FAO genes. ConclusionsOur data identify Egr1 as a novel modulator of NAFLD and a potential target for NAFLD intervention. Impact and ImplicationsNon-alcoholic fatty liver disease (NAFLD) precedes cirrhosis and hepatocellular carcinoma. In this paper, we describe a novel mechanism whereby early growth response 1 (Egr1), a transcription factor, contributes to NAFLD pathogenesis by regulating fatty acid oxidation. Our data provide novel insights and translational potential for NAFLD intervention.

Plasticity in airway smooth muscle differentiation during mouse lung development.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

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.

Myocardin-related transcription factor A, regulated by serum response factor, contributes to diabetic cardiomyopathy in mice

AimsDiabetic cardiomyopathy is a significant contributor to the global pandemic of heart failure. In the present study we investigated the involvement of myocardin-related transcription factor A (MRTF-A), a transcriptional regulator, in this process. Materials and methodsDiabetic cardiomyopathy was induced in mice by feeding with a high-fat diet (HFD) or streptozotocin (STZ) injection. Key findingsWe report that MRTF-A was up-regulated in the hearts of mice with diabetic cardiomyopathy. MRTF-A expression was also up-regulated by treatment with palmitate in cultured cardiomyocytes in vitro. Mechanistically, serum response factor (SRF) bound to the MRTF-A gene promoter and activated MRTF-A transcription in response to pro-diabetic stimuli. Knockdown of SRF abrogated MRTF-A induction in cardiomyocytes treated with palmitate. When cardiomyocytes conditional MRTF-A knockout mice (MRTF-A CKO) and wild type (WT) mice were placed on an HFD to induce diabetic cardiomyopathy, it was found that the CKO mice and the WT mice displayed comparable metabolic parameters including body weight, blood insulin concentration, blood cholesterol concentration, and glucose tolerance. However, both systolic and diastolic cardiac function were exacerbated by MRTF-A deletion in the heart. SignificanceThese data suggest that MRTF-A up-regulation might serve as an important compensatory mechanism to safeguard the deterioration of cardiac function during diabetic cardiomyopathy.

Critical Role of LMCD1 in Promoting Profibrotic Characteristics of Lung Myofibroblasts in Experimental and Scleroderma-Associated Lung Fibrosis.

Interstitial lung disease (ILD) is a serious complication and leading cause of mortality in patients with systemic sclerosis (SSc). In this study, we explored the role of LIM and cysteine-rich domains protein 1 (LMCD1) as a novel factor in the pathogenesis of SSc-related ILD (SSc-ILD). The expression and effects of LMCD1 were studied in lung tissue samples and fibroblasts from SSc-ILD patients and control subjects as well as in lung tissue samples from animal models. LMCD1 was consistently elevated in lung tissue samples and in fibroblasts isolated from SSc-ILD patients as compared to controls. Additionally, LMCD1 was found to be highly expressed in the lung in the fibroblast-specific protein (FSP)-driven, constitutively active transforming growth factor β receptor type I (TGFβR1) transgenic mouse model of ILD and the bleomycin-induced mouse model of ILD. In lung fibroblasts from SSc-ILD patients, LMCD1 is an essential factor for the TGFβ-induced generation of type I collagen, fibronectin, and α-smooth muscle actin (α-SMA). Depletion of LMCD1 by small interfering RNA reduced the expression of extracellular matrix proteins and lowered transcriptional activity and expression of α-SMA, as well as decreased the proliferation and contractile activity of SSc-ILD lung fibroblasts. In dense fibrotic areas of affected lung tissue, lung LMCD1 colocalized with α-SMA. In cultured scleroderma lung fibroblasts, LMCD1 colocalized and interacted with serum response factor which mediates LMCD1-induced contractile activity of lung fibroblasts. Our study identifies LMCD1 as a profibrotic molecule contributing to the activation of myofibroblasts and the persistent fibroproliferation observed in SSc-ILD. Thus, LMCD1 may be a potential novel therapeutic target for patients with SSc-ILD.

The β-Blocker Carvedilol Prevents Benzo(a)pyrene-Induced Lung Toxicity, Inflammation and Carcinogenesis.

The current study evaluated the effects of the β-blocker carvedilol on benzo(a)pyrene (B(a)P) and its active metabolite benzo(a)pyrene diol epoxide (BPDE)-induced lung toxicity, inflammation and carcinogenesis and explored the potential mechanisms. Carvedilol blocked the BPDE-induced malignant transformation of human bronchial epithelial cells BEAS-2B. In BEAS-2B cells, B(a)P strongly activated ELK-1, a transcription factor regulating serum response element (SRE) signaling, which was attenuated by carvedilol. Carvedilol also inhibited the B(a)P-induced AhR/xenobiotic responsive element (XRE) and mRNA expression of CYP1A1 and attenuated B(a)P-induced NF-κB activation. In a B(a)P-induced acute lung toxicity model in CD-1/IGS mice, pretreatment with carvedilol for 7 days before B(a)P exposure effectively inhibited the B(a)P-induced plasma levels of lactate dehydrogenase and malondialdehyde, inflammatory cell infiltration and histopathologic abnormalities in the lung, and upregulated the expression of GADD45α, caspase-3 and COX-2 in the lung. In a B(a)P-induced lung carcinogenesis model in A/J mice, carvedilol treatment for 20 weeks did not affect body weight but significantly attenuated tumor multiplicity and volume. These data reveal a previously unexplored role of carvedilol in preventing B(a)P-induced lung inflammation and carcinogenesis by inhibiting the cross-talk of the oncogenic transcription factors ELK-1, AhR and NF-κB.

Empagliflozin inhibits excessive autophagy through the AMPK/GSK3β signalling pathway in diabetic cardiomyopathy.

Sodium-glucose cotransporter 2 inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and non-diabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy. Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day), had fewer apoptotic cells (4.9 ± 2.1 vs. 1 ± 0.5 TUNEL-positive cells %, P < 0.05), less senescence (10.1 ± 2 vs. 7.9 ± 1.2 β-gal positivity/tissue area, P < 0.05), fibrosis (0.2 ± 0.05 vs. 0.15 ± 0.06, P < 0.05 fibrotic area/tissue area), autophagy (7.9 ± 0.05 vs. 2.3 ± 0.6 fluorescence intensity/total area, P < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a down-regulation of the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to the modulation of cardiomyogenic transcription factors, we analysed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice [0.5 ± 0.01 densitometric units (DU)], non-diabetic mice treated with EMPA (2.2 ± 0.01 DU, P < 0.01) and diabetic mice treated with EMPA (2.0 ± 0.1 DU, P < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 ± 0.1 vs. 2.2 ± 0.01 target protein/β-actin ratio, P < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer glycogen synthase kinase 3 beta (GSK3β), leading to reactivation of cardiomyogenic transcription factors. Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3β signalling pathway in the context of diabetes.

Myofibroblast-specific inhibition of the Rho kinase-MRTF-SRF pathway using nanotechnology for the prevention of pulmonary fibrosis.

Pulmonary fibrosis is characterized by the accumulation of myofibroblasts in the lung and progressive tissue scarring. Fibroblasts exist across a spectrum of states, from quiescence in health to activated myofibroblasts in the setting of injury. Highly activated myofibroblasts have a critical role in the establishment of fibrosis as the predominant source of type 1 collagen and profibrotic mediators. Myofibroblasts are also highly contractile cells and can alter lung biomechanical properties through tissue contraction. Inhibiting signaling pathways involved in myofibroblast activation could therefore have significant therapeutic value. One of the ways myofibroblast activation occurs is through activation of the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) pathway, which signals through intracellular actin polymerization. However, concerns surrounding the pleiotropic and ubiquitous nature of these signaling pathways have limited the translation of inhibitory drugs. Herein, we demonstrate a novel therapeutic antifibrotic strategy using myofibroblast-targeted nanoparticles containing a MTRF/SRF pathway inhibitor (CCG-1423), which has been shown to block myofibroblast activation in vitro. Myofibroblasts were preferentially targeted via the angiotensin 2 receptor, which has been shown to be selectively upregulated in animal and human studies. These nanoparticles were nontoxic and accumulated in lung myofibroblasts in the bleomycin-induced mouse model of pulmonary fibrosis, reducing the number of these activated cells and their production of profibrotic mediators. Ultimately, in a murine model of lung fibrosis, a single injection of these drugs containing targeted nanoagents reduced fibrosis as compared with control mice. This approach has the potential to deliver personalized therapy by precisely targeting signaling pathways in a cell-specific manner, allowing increased efficacy with reduced deleterious off-target effects.

P. gingivalis-LPS Induces Mitochondrial Dysfunction Mediated by Neuroinflammation through Oxidative Stress.

Porphyromonas gingivalis (P. gingivalis), a key pathogen in periodontitis, is associated with neuroinflammation. Periodontal disease increases with age; 70.1% of adults 65 years and older have periodontal problems. However, the P. gingivalis- lipopolysaccharide (LPS)induced mitochondrial dysfunction in neurodegenerative diseases remains elusive. In this study, we investigated the possible role of P. gingivalis-LPS in mitochondrial dysfunction during neurodegeneration. We found that P. gingivalis-LPS treatment activated toll-like receptor (TLR) 4 signaling and upregulated the expression of Alzheimer's disease-related dementia and neuroinflammatory markers. Furthermore, the LPS treatment significantly exacerbated the production of reactive oxygen species and reduced the mitochondrial membrane potential. Our study highlighted the pivotal role of P. gingivalis-LPS in the repression of serum response factor (SRF) and its co-factor p49/STRAP that regulate the actin cytoskeleton. The LPS treatment repressed the genes involved in mitochondrial function and biogenesis. P. gingivalis-LPS negatively altered oxidative phosphorylation and glycolysis and reduced total adenosine triphosphate (ATP) production. Additionally, it specifically altered the mitochondrial functions in complexes I, II, and IV of the mitochondrial electron transport chain. Thus, it is conceivable that P. gingivalis-LPS causes mitochondrial dysfunction through oxidative stress and inflammatory events in neurodegenerative diseases.

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.

Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition.

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Role of CRP2-MRTF interaction in functions of myofibroblasts

Inflammatory response induces phenotypic modulation of fibroblasts into myofibroblasts. Although transforming growth factor-βs (TGF-βs) evoke such transition, the details of the mechanism are still unknown. Here, we report that a LIM domain protein, cysteine-and glycine-rich protein 2 (CSRP2 [CRP2]) plays a vital role in the functional expression profile in myofibroblasts and cancer-associated fibroblasts (CAFs). Knock-down of CRP2 severely inhibits the expression of smooth muscle cell (SMC) genes, cell motility, and CAF-mediated collective invasion of epidermoid carcinoma. We elucidate the following molecular bases: CRP2 directly binds to myocardin-related transcription factors (MRTF-A/B [MRTFs]) and serum response factor (SRF) and stabilizes the MRTF/SRF/CArG-box complex to activate SMC gene expression. Furthermore, a three-dimensional structural analysis of CRP2 identifies the amino acids required for the CRP2-MRTF-A interaction. Polar amino acids in the C-terminal half (serine-152, glutamate-154, serine-155, threonine-156, threonine-157, and threonine-159 in human CRP2) are responsible for direct binding to MRTF-A. On the other hand, hydrophobic amino acids outside the consensus sequence of the LIM domain (tryptophan-139, phenylalanine-144, leucine-153, and leucine-158 in human CRP2) play a role in stabilizing the unique structure of the LIM domain.Key words: CRP2, 3D structure, myocardin-related transcription factor, myofibroblast, cancer-associated fibroblasts.

Abstract 1959: A phosphorylation mimic at the Gα12 N-terminus inhibits palmitoylation and signaling to serum response factor

Abstract 1959: A phosphorylation mimic at the Gα12 N-terminus inhibits palmitoylation and signaling to serum response factor