A myocardin-adjacent lncRNA balances SRF-dependent gene transcription in the heart.

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.

SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination.

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses.

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in transcription of heat shock protein (HSP)-encoding genes in fly ovaries, but the mechanisms remain unclear. Here, we demonstrate that, in mammalian cells, MRTFs are dispensable for gene induction of HSP-encoding genes. However, the widely used small-molecule inhibitors of the MRTF-SRF transcription pathway, derived from CCG-1423, also efficiently inhibit gene transcription of HSP-encoding genes in both fly and mammalian cells in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF-SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

Sall1 and Sall4 cooperatively interact with Myocd and SRF to promote cardiomyocyte proliferation by regulating CDK and cyclin genes.

Sall1 and Sall4 (Sall1/4), zinc-finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4 functionally inhibited mice by forced expression of the truncated form of Sall4 (ΔSall4) in the heart. The ΔSall4-overexpression mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from the SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were reduced in ΔSall4-overexpression mice. RNA-sequencing data showed that Sall1/4 act upstream of the cyclin-dependent kinase (CDK) and cyclin genes, and of key transcription factor genes for the development of compact cardiomyocytes, including myocardin (Myocd) and serum response factor (Srf). In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with SRF, and directly bind to the upstream regulatory regions of the CDK and cyclin genes (Cdk1 and Ccnb1). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of CDK and cyclin genes that interact with Myocd and SRF.

Brain-derived neurotrophic factor (BDNF) downregulates mRNA levels of suppressor of cancer cell invasion (SCAI) variants in cortical neurons.

Suppressor of cancer cell invasion (SCAI) acts as a transcriptional repressor of serum response factor (SRF)-mediated gene expression by binding to megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which is an SRF transcriptional coactivator. Growing evidence suggests that SCAI is a negative regulator of neuronal morphology, whereas MKL2/MRTFB is a positive regulator. The mRNA expression of SCAI is downregulated during brain development, suggesting that a reduction in SCAI contributes to the reduced suppression of SRF-mediated gene induction, thus increasing dendritic complexity and developing neuronal circuits. In the present study, we hypothesized that brain-derived neurotrophic factor (BDNF), which is important for neuronal plasticity and development, might alter SCAI mRNA levels. We therefore investigated the effects of BDNF on SCAI mRNA levels in primary cultured cortical neurons. Furthermore, because alternative splicing generates several SCAI variants in the brain, we measured SCAI variant mRNA after BDNF stimulation. Both SCAI variant 1 and total SCAI mRNA expression levels were downregulated by BDNF. Moreover, the extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) pathway was involved in the BDNF-mediated decrease in SCAI mRNA expression. Our findings provide insights into the molecular mechanism underlying a neurotrophic factor switch for the repressive transcriptional complex that includes SCAI.

ERK-Induced Activation of TCF Family of SRF Cofactors Initiates a Chromatin Modification Cascade Associated with Transcription.

We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.

LncRNA CARMN inhibits abdominal aortic aneurysm formation and vascular smooth muscle cell phenotypic transformation by interacting with SRF.

Phenotypic transformation of vascular smooth muscle cells (VSMCs) plays a crucial role in abdominal aortic aneurysm (AAA) formation. CARMN, a highly conserved, VSMC-enriched long noncoding RNA (lncRNA), is integral in orchestrating various vascular pathologies by modulating the phenotypic dynamics of VSMCs. The influence of CARMN on AAA formation, particularly its mechanisms, remains enigmatic. Our research, employing single-cell and bulk RNA sequencing, has uncovered a significant suppression of CARMN in AAA specimens, which correlates strongly with the contractile function of VSMCs. This reduced expression of CARMN was consistent in both 7- and 14-day porcine pancreatic elastase (PPE)-induced mouse models of AAA and in human clinical cases. Functional analyses disclosed that the diminution of CARMN exacerbated PPE-precipitated AAA formation, whereas its augmentation conferred protection against such formation. Mechanistically, we found CARMN's capacity to bind with SRF, thereby amplifying its role in driving the transcription of VSMC marker genes. In addition, our findings indicate an enhancement in CAMRN transcription, facilitated by the binding of NRF2 to its promoter region. Our study indicated that CARMN plays a protective role in preventing AAA formation and restrains the phenotypic transformation of VSMC through its interaction with SRF. Additionally, we observed that the expression of CARMN is augmented by NRF2 binding to its promoter region. These findings suggest the potential of CARMN as a viable therapeutic target in the treatment of AAA.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease. METHODS: Wire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype. RESULTS: Phenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation. CONCLUSIONS: The present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis.

Mutual dependence of the MRTF-SRF and YAP-TEAD pathways in cancer-associated fibroblasts is indirect and mediated by cytoskeletal dynamics.

Both the MRTF-SRF and the YAP-TEAD transcriptional regulatory networks respond to extracellular signals and mechanical stimuli. We show that the MRTF-SRF pathway is activated in cancer-associated fibroblasts (CAFs). The MRTFs are required in addition to the YAP pathway for CAF contractile and proinvasive properties. We compared MRTF-SRF and YAP-TEAD target gene sets and identified genes directly regulated by one pathway, the other, or both. Nevertheless, the two pathways exhibit mutual dependence. In CAFs, expression of direct MRTF-SRF genomic targets is also dependent on YAP-TEAD activity, and, conversely, YAP-TEAD target gene expression is also dependent on MRTF-SRF signaling. In normal fibroblasts, expression of activated MRTF derivatives activates YAP, while activated YAP derivatives activate MRTF. Cross-talk between the pathways requires recruitment of MRTF and YAP to DNA via their respective DNA-binding partners (SRF and TEAD) and is therefore indirect, arising as a consequence of activation of their target genes. In both CAFs and normal fibroblasts, we found that YAP-TEAD activity is sensitive to MRTF-SRF-induced contractility, while MRTF-SRF signaling responds to YAP-TEAD-dependent TGFß signaling. Thus, the MRF-SRF and YAP-TEAD pathways interact indirectly through their ability to control cytoskeletal dynamics.

Myocardin regulates fibronectin expression and secretion from human pleural mesothelial cells.

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-ß induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-ß stimulation at nucleus, suggesting that TGF-ß facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.

TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity.

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.

Lysine acetyltransferase 14 mediates TGF-ß-induced fibrosis in ovarian endometrioma via co-operation with serum response factor.

BACKGROUND: Fibrogenesis within ovarian endometrioma (endometrioma), mainly induced by transforming growth factor-ß (TGF-ß), is characterized by myofibroblast over-activation and excessive extracellular matrix (ECM) deposition, contributing to endometrioma-associated symptoms such as infertility by impairing ovarian reserve and oocyte quality. However, the precise molecular mechanisms that underpin the endometrioma- associated fibrosis progression induced by TGF-ß remain poorly understood. METHODS: The expression level of lysine acetyltransferase 14 (KAT14) was validated in endometrium biopsies from patients with endometrioma and healthy controls, and the transcription level of KAT14 was further confirmed by analyzing a published single-cell transcriptome (scRNA-seq) dataset of endometriosis. We used overexpression, knockout, and knockdown approaches in immortalized human endometrial stromal cells (HESCs) or human primary ectopic endometrial stromal cells (EcESCs) to determine the role of KAT14 in TGF-ß-induced fibrosis. Furthermore, an adeno-associated virus (AAV) carrying KAT14-shRNA was used in an endometriosis mice model to assess the role of KAT14 in vivo. RESULTS: KAT14 was upregulated in ectopic lesions from endometrioma patients and predominantly expressed in activated fibroblasts. In vitro studies showed that KAT14 overexpression significantly promoted a TGF-ß-induced profibrotic response in endometrial stromal cells, while KAT14 silencing showed adverse effects that could be rescued by KAT14 re-enhancement. In vivo, Kat14 knockdown ameliorated fibrosis in the ectopic lesions of the endometriosis mouse model. Mechanistically, we showed that KAT14 directly interacted with serum response factor (SRF) to promote the expression of α-smooth muscle actin (α-SMA) by increasing histone H4 acetylation at promoter regions; this is necessary for TGF-ß-induced ECM production and myofibroblast differentiation. In addition, the knockdown or pharmacological inhibition of SRF significantly attenuated KAT14-mediating profibrotic effects under TGF-ß treatment. Notably, the KAT14/SRF complex was abundant in endometrioma samples and positively correlated with α-SMA expression, further supporting the key role of KAT14/SRF complex in the progression of endometrioma-associated fibrogenesis. CONCLUSION: Our results shed light on KAT14 as a key effector of TGF-ß-induced ECM production and myofibroblast differentiation in EcESCs by promoting histone H4 acetylation via co-operating with SRF, representing a potential therapeutic target for endometrioma-associated fibrosis.

Serum Response Factor Reduces Gene Expression Noise and Confers Cell State Stability.

The role of serum response factor (Srf), a central mediator of actin dynamics and mechanical signaling, in cell identity regulation is debated to be either a stabilizer or a destabilizer. We investigated the role of Srf in cell fate stability using mouse pluripotent stem cells. Despite the fact that serum-containing cultures yield heterogeneous gene expression, deletion of Srf in mouse pluripotent stem cells leads to further exacerbated cell state heterogeneity. The exaggerated heterogeneity is detectible not only as increased lineage priming but also as the developmentally earlier 2C-like cell state. Thus, pluripotent cells explore more variety of cellular states in both directions of development surrounding naïve pluripotency, a behavior that is constrained by Srf. These results support that Srf functions as a cell state stabilizer, providing rationale for its functional modulation in cell fate intervention and engineering.

SRF Co-factors Control the Balance between Cell Proliferation and Contractility.

The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression.

Periostin regulates lysyl oxidase through ERK1/2 MAPK-dependent serum response factor in activated cardiac fibroblasts.

Collagen crosslinking, mediated by lysyl oxidase, is an adaptive mechanism of the cardiac repair process initiated by cardiac fibroblasts postmyocardial injury. However, excessive crosslinking leads to cardiac wall stiffening, which impairs the contractile properties of the left ventricle and leads to heart failure. In this study, we investigated the role of periostin, a matricellular protein, in the regulation of lysyl oxidase in cardiac fibroblasts in response to angiotensin II and TGFß1. Our results indicated that periostin silencing abolished the angiotensin II and TGFß1-mediated upregulation of lysyl oxidase. Furthermore, the attenuation of periostin expression resulted in a notable reduction in the activity of lysyl oxidase. Downstream of periostin, ERK1/2 MAPK signaling was found to be activated, which in turn transcriptionally upregulates the serum response factor to facilitate the enhanced expression of lysyl oxidase. The periostin-lysyl oxidase association was also positively correlated in an in vivo rat model of myocardial infarction. The expression of periostin and lysyl oxidase was upregulated in the collagen-rich fibrotic scar tissue of the left ventricle. Remarkably, echocardiography data showed a reduction in the left ventricular wall movement, ejection fraction, and fractional shortening, indicative of enhanced stiffening of the cardiac wall. These findings shed light on the mechanistic role of periostin in the collagen crosslinking initiated by activated cardiac fibroblasts. Our findings signify periostin as a possible therapeutic target to reduce excessive collagen crosslinking that contributes to the structural remodeling associated with heart failure.

Cardiac Transcription Factors and Regulatory Networks.

Cardiac development is a fine-tuned process governed by complex transcriptional networks, in which transcription factors (TFs) interact with other regulatory layers. In this chapter, we introduce the core cardiac TFs including Gata, Hand, Nkx2, Mef2, Srf, and Tbx. These factors regulate each other's expression and can also act in a combinatorial manner on their downstream targets. Their disruption leads to various cardiac phenotypes in mice, and mutations in humans have been associated with congenital heart defects. In the second part of the chapter, we discuss different levels of regulation including cis-regulatory elements, chromatin structure, and microRNAs, which can interact with transcription factors, modulate their function, or are downstream targets. Finally, examples of disturbances of the cardiac regulatory network leading to congenital heart diseases in human are provided.

Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling.

The paucity of knowledge about cardiomyocyte maturation is a major bottleneck in cardiac regenerative medicine. In development, cardiomyocyte maturation is characterized by orchestrated structural, transcriptional, and functional specializations that occur mainly at the perinatal stage. Sarcomeres are the key cytoskeletal structures that regulate the ultrastructural maturation of other organelles, but whether sarcomeres modulate the signal transduction pathways that are essential for cardiomyocyte maturation remains unclear. To address this question, here we generated mice with cardiomyocyte-specific, mosaic, and hypomorphic mutations of α-actinin-2 (Actn2) to study the cell-autonomous roles of sarcomeres in postnatal cardiomyocyte maturation. Actn2 mutation resulted in defective structural maturation of transverse-tubules and mitochondria. In addition, Actn2 mutation triggered transcriptional dysregulation, including abnormal expression of key sarcomeric and mitochondrial genes, and profound impairment of the normal progression of maturational gene expression. Mechanistically, the transcriptional changes in Actn2 mutant cardiomyocytes strongly correlated with those in cardiomyocytes deleted of serum response factor (SRF), a critical transcription factor that regulates cardiomyocyte maturation. Actn2 mutation increased the monomeric form of cardiac α-actin, which interacted with the SRF cofactor MRTFA and perturbed its nuclear localization. Overexpression of a dominant-negative MRTFA mutant was sufficient to recapitulate the morphological and transcriptional defects in Actn2 and Srf mutant cardiomyocytes. Together, these data indicate that Actn2-based sarcomere organization regulates structural and transcriptional maturation of cardiomyocytes through MRTF-SRF signaling.

Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM).

The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ∼3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3' untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ∼3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF-myocardin-related TF (MRTF) activity bouts in proliferating cells.

The Rho guanine nucleotide exchange factor PLEKHG1 is activated by interaction with and phosphorylation by Src family kinase member FYN.

Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.

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 upregulated in calcific areas. We further demonstrated TCF21 expression was increased 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-1ß 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.