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

Abstract Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular regulation of megakaryocytopoiesis. MKL1 is part of the fusion protein formed by the t (1; 22) translocation, which is found uniquely in Acute Megakaryoblastic Leukemia (AMKL). The translocation fuses the RBM15 (also known as OTT) gene on chromosome 1 with the MKL1 (also known as MAL) gene on chromosome 22. Previous studies in muscle cells show that MKL1 is a positive cofactor for the transcription factor serum response factor (SRF), and works via the Rho-A pathway to turn on immediate early genes and muscle specific genes. Using qRT-PCR we show that MKL1 mRNA is markedly up-regulated during megakaryocyte (MK) differentiation of primary murine bone marrow and fetal liver cells. When we overexpress MKL1 in the human erythroleukemia (HEL) cell line and differentiate the cells to become MK by phorbol ester (TPA), there is far greater MK differentiation than in control HEL cells. Via analysis of Wright-Geimsa stained cytospins, MKL1 overexpression increases the average percentage of mature MK from 26% to 48%. Using flow cytometry, platelet glycoprotein V positive cells increase from 14% to 43% on average. The percentage of cells with greater than 4N ploidy increases from 13% to 34%. In order to assess the mechanisms by which MKL1 promotes MK differentiation, we tested whether SRF expression was required for the effects of MKL1. The stimulatory effects of MKL1 are strongly abrogated when cells are transfected with siRNA against SRF, proving that MKL1 acts via SRF to stimulate MK differentiation. Interestingly, SRF siRNA also causes a statistically significant decrease in ploidy in control cells stimulated with TPA. By microarray analyses using both Affymetrix and Illumina platforms, enforced MKL1 expression upregulates many cytoskeletal genes and adhesion molecules, enhances the expression of platelet specific genes such as glycoprotein V (consistent with the FACS data), and accelerates the loss of expression of genes associated with erythropoiesis, such as erythrocyte membrane protein band 4.2. These data indicate that MKL1 enhances MK differentiation by promoting endoduplication as well as increasing expression of platelet-specific genes and of multiple cytoskeletal proteins required for proplatelet formation.

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Interaction of Serum Response Factor (SRF) with the Elk-1 B Box Inhibits RhoA-Actin Signaling to SRF and Potentiates Transcriptional Activation by Elk-1

Molecular and Cellular Biology ◽

10.1128/mcb.22.20.7083-7092.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 7083-7092 ◽

Cited By ~ 40

Author(s):

Kasumi Murai ◽

Richard Treisman

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Rho Gtpases ◽

Target Genes ◽

Serum Response Factor ◽

Relative Sensitivity ◽

Actin Dynamics ◽

Physical Interaction ◽

Response Factor ◽

Serum Response

ABSTRACT Serum response factor (SRF) is a transcription factor which regulates many immediate-early genes. Rho GTPases regulate SRF activity through changes in actin dynamics, but some SRF target genes, such as c-fos, are insensitive to this pathway. At the c-fos promoter, SRF recruits members of the ternary complex factor (TCF) family of Ets domain proteins through interactions with the TCF B-box region. Analysis of c-fos promoter mutations demonstrates that the TCF and ATF/AP1 sites adjoining the SRF binding site inhibit activation of the promoter by RhoA-actin signaling. The presence of the TCF binding site is sufficient for inhibition, and experiments with an altered-specificity Elk-1 derivative demonstrate that inhibition can be mediated by the Elk-1 TCF. Using Elk-1 fusion proteins that can bind DNA autonomously, we show that inhibition of RhoA-actin signaling requires physical interaction between the Elk-1 B box and SRF. These results account for the insensitivity of c-fos to RhoA-actin signaling. Interaction of the B box with SRF also potentiates transcriptional activation by the Elk-1 C-terminal activation domain. Combinatorial interactions between SRF and TCF proteins are thus likely to play an important role in determining the relative sensitivity of SRF target genes to Ras- and Rho-controlled signal transduction pathways.

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Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus

AJP Cell Physiology ◽

10.1152/ajpcell.00386.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. C70-C81 ◽

Cited By ~ 298

Author(s):

Joseph M. Miano ◽

Xiaochun Long ◽

Keigi Fujiwara

Keyword(s):

Transcription Factor ◽

Actin Cytoskeleton ◽

Target Genes ◽

Serum Response Factor ◽

Cellular Growth ◽

Cellular Migration ◽

Actin Dynamics ◽

Response Factor ◽

Master Regulator ◽

Serum Response

Serum response factor (SRF) is a highly conserved and widely expressed, single copy transcription factor that theoretically binds up to 1,216 permutations of a 10-base pair cis element known as the CArG box. SRF-binding sites were defined initially in growth-related genes. Gene inactivation or knockdown studies in species ranging from unicellular eukaryotes to mice have consistently shown loss of SRF to be incompatible with life. However, rather than being critical for proliferation and growth, these genetic studies point to a crucial role for SRF in cellular migration and normal actin cytoskeleton and contractile biology. In fact, recent genomic studies reveal nearly half of the >200 SRF target genes encoding proteins with functions related to actin dynamics, lamellipodial/filopodial formation, integrin-cytoskeletal coupling, myofibrillogenesis, and muscle contraction. SRF has therefore emerged as a dispensable transcription factor for cellular growth but an absolutely essential orchestrator of actin cytoskeleton and contractile homeostasis. This review summarizes the recent genomic and genetic analyses of CArG-SRF that support its role as an ancient, master regulator of the actin cytoskeleton and contractile machinery.

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Megakaryoblastic Leukemia 1, a Potent Transcriptional Coactivator for Serum Response Factor (SRF), Is Required for Serum Induction of SRF Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.23.18.6597-6608.2003 ◽

2003 ◽

Vol 23 (18) ◽

pp. 6597-6608 ◽

Cited By ~ 207

Author(s):

Bo Cen ◽

Ahalya Selvaraj ◽

Rebecca C. Burgess ◽

Johann K. Hitzler ◽

Zhigui Ma ◽

...

Keyword(s):

Target Genes ◽

Serum Response Factor ◽

Gene Activation ◽

Reporter Genes ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Related Factor ◽

Response Factor ◽

Megakaryoblastic Leukemia ◽

Serum Response

ABSTRACT Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

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Target Gene-Specific Modulation of Myocardin Activity by GATA Transcription Factors

Molecular and Cellular Biology ◽

10.1128/mcb.24.19.8519-8528.2004 ◽

2004 ◽

Vol 24 (19) ◽

pp. 8519-8528 ◽

Cited By ~ 44

Author(s):

Jiyeon Oh ◽

Zhigao Wang ◽

Da-Zhi Wang ◽

Ching-Ling Lien ◽

Weibing Xing ◽

...

Keyword(s):

Transcription Factors ◽

Target Gene ◽

Target Genes ◽

Serum Response Factor ◽

Fine Tuning ◽

Physical Interaction ◽

Response Factor ◽

Muscle Gene Expression ◽

Gata Transcription Factors ◽

Serum Response

ABSTRACT Myocardin is a transcriptional coactivator that regulates cardiac and smooth muscle gene expression by associating with serum response factor. We show that GATA transcription factors can either stimulate or suppress the transcriptional activity of myocardin, depending on the target gene. Modulation of myocardin activity by GATA4 is mediated by the physical interaction of myocardin with the DNA binding domain of GATA4 but does not require binding of GATA4 to DNA. Paradoxically, the transcription activation domain of GATA4 is dispensable for the stimulatory effect of GATA4 on myocardin activity but is required for repression of myocardin activity. The ability of GATA transcription factors to modulate myocardin activity provides a potential mechanism for fine tuning the expression of serum response factor target genes in a gene-specific manner.

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