Serum response factor (SRF) binds a 1216-fold degenerate cis element known as the CArG box. CArG boxes are found primarily in muscle- and growth-factor-associated genes although the full spectrum of functional CArG elements in the genome (the CArGome) has yet to be defined. Here we describe a genome-wide screen to further define the functional mammalian CArGome. A computational approach involving comparative genomic analyses of human and mouse orthologous genes uncovered >100 hypothetical SRF-dependent genes, including 10 previously identified SRF targets, harboring a conserved CArG element within 4000 bp of the annotated transcription start site (TSS). We PCR-cloned 89 hypothetical SRF targets and subjected each of them to at least two of several validations including luciferase reporter, gel shift, chromatin immunoprecipitation, and mRNA expression following RNAi knockdown of SRF; 60/89 (67%) of the targets were validated. Interestingly, 26 of the validated SRF target genes encode for cytoskeletal/contractile or adhesion proteins. RNAi knockdown of SRF diminishes expression of several SRF-dependent cytoskeletal genes and elicits an attending perturbation in the cytoarchitecture of both human and rodent cells. These data illustrate the power of integrating existing algorithms to interrogate the genome in a relatively unbiased fashion for cis-regulatory element discovery. In this manner, we have further expanded the mammalian CArGome with the discovery of an array of cyto-contractile genes that coordinate normal cytoskeletal homeostasis. We suggest one function of SRF is that of an ancient master regulator of the actin cytoskeleton.

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Amyloid β-peptide (Aβ) deposition in cerebral vessels contributes to cerebral amyloid angiopathy (CAA) in Alzheimer’s disease (AD). Here, we report that in AD patients and two mouse models of AD, overexpression of serum response factor (SRF) and myocardin (MYOCD) in cerebral vascular smooth muscle cells (VSMCs) generates an Aβ non-clearing VSMC phenotype through transactivation of sterol regulatory element binding protein-2, which downregulates low density lipoprotein receptor-related protein-1, a key Aβ clearance receptor. Hypoxia stimulated SRF/MYOCD expression in human cerebral VSMCs and in animal models of AD. We suggest that SRF and MYOCD function as a transcriptional switch, controlling Aβ cerebrovascular clearance and progression of AD.

Abstract-Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)-induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondriaspecific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities. Key Words: thioredoxin Ⅲ ASK1 Ⅲ mitochondria Ⅲ TNF-␣ Ⅲ apoptosis T hioredoxins (Trx) are cellular redox enzymes that have multiple functions in regulation of cell growth, apoptosis, and activation. 1 Trx contains two redox-active cysteine residues in its catalytic center with a consensus amino acid sequence: cys-gly-pro-cys. Trx exists either in a reduced dithiol form or in an oxidized form. It participates in redox reactions by reversible oxidation of its active center dithiol to disulfide and catalyzes dithio-disulfide exchange reactions involving many thiol-dependent processes. 1 Thus, Trx system is considered to constitute an endogenous antioxidant system in addition to the glutathione and superoxide dismutase systems. Two isoforms of Trx have been identified in mammalian cells: cytosolic and mitochondrial Trx (Trx1 and Trx2, respectively). Trx1 and Trx2 are encoded by distinct nuclear genes and Trx2 contains a mitochondrial targeting signal peptide. 2-4 The mitochondrion is the major source of ROS generated during physiological respiration and pathological conditions during inflammation in response to cytokines. 5 Therefore, the mitochondrial antioxidant systems including Trx2, Trx2 reductase, mitochondrial Trx peroxidas...

Cerebral angiopathy contributes to cognitive decline and dementia in Alzheimer's disease (AD) through cerebral blood flow (CBF) reductions and dysregulation. We report vascular smooth muscle cells (VSMC) in small pial and intracerebral arteries, which are critical for CBF regulation, express in AD high levels of serum response factor (SRF) and myocardin (MYOCD), two interacting transcription factors that orchestrate a VSMC-differentiated phenotype. Consistent with this finding, AD VSMC overexpressed several SRF-MYOCD-regulated contractile proteins and exhibited a hypercontractile phenotype. MYOCD overexpression in control human cerebral VSMC induced an AD-like hypercontractile phenotype and diminished both endothelial-dependent and -independent relaxation in the mouse aorta ex vivo. In contrast, silencing SRF normalized contractile protein content and reversed a hypercontractile phenotype in AD VSMC. MYOCD in vivo gene transfer to mouse pial arteries increased contractile protein content and diminished CBF responses produced by brain activation in wild-type mice and in two AD models, the Dutch/Iowa/Swedish triple mutant human amyloid beta-peptide (Abeta)-precursor protein (APP)- expressing mice and APPsw(+/-) mice. Silencing Srf had the opposite effect. Expression of SRF did not change in VSMC subjected to Alzheimer's neurotoxin, Abeta. Thus, SRF-MYOCD overexpression in small cerebral arteries appears to initiate independently of Abeta a pathogenic pathway mediating arterial hypercontractility and CBF dysregulation, which are associated with Alzheimer's dementia.

A Kinase Anchoring Protein 12 (AKAP12; also known as src-suppressed C kinase substrate (SSeCKS) and Gravin) is a multivalent anchoring protein with tumor suppressor activity. Although expression of AKAP12 has been examined in a number of contexts, its expression control remains to be elucidated. Herein, we characterize the genomic organization of the AKAP12 locus, its regulatory regions, and the spatial distribution of the proteins encoded by the AKAP12 gene. Using comparative genomics and various wet-lab assays, we show that the AKAP12 locus is organized as three separate transcription units that are governed by non-redundant promoters coordinating distinct tissue expression profiles. The proteins encoded by the three AKAP12 isoforms (designated ␣, ␤, and ␥) share >95% amino acid sequence identity but differ at their N termini. Analysis of the targeting of each isoform reveals distinct spatial distribution profiles. An N-terminal myristoylation motif present in AKAP12␣ is shown to be necessary and sufficient for targeted expression of this AKAP12 isoform to the endoplasmic reticulum, a novel subcellular compartment for AKAP12. Our results demonstrate heretofore unrecognized complexity within the AKAP12 locus and suggest a mechanism for genetic control of signaling specificity through distinct regulation of alternately targeted anchoring protein isoforms.

Retinoids block smooth muscle cell (SMC) proliferation and attenuate neointimal formation after vascular injury, presumably through retinoid receptor-mediated changes in gene expression. To identify target genes in SMC whose encoded proteins could contribute to such favorable biological effects, we performed a subtractive screen for retinoid-inducible genes in cultured SMC. Here, we report on the cloning and initial characterization of a novel retinoid-inducible serine carboxypeptidase (RISC). Expression of RISC is low in cultured SMC but progressively increases over a 5-day time-course treatment with all-trans-retinoic acid. A near full-length rat RISC cDNA was cloned and found to have a 452-amino acid open reading frame containing an aminoterminal signal sequence, followed by several conserved domains comprising the catalytic triad common to members of the serine carboxypeptidase family. In vitro transcription and translation experiments showed that the rat RISC cDNA generates a ϳ51-kDa protein. Confocal immunofluorescence microscopy of COS-7 cells transiently transfected with a RISC-His tag plasmid revealed cytosolic localization of the fusion protein.Western blotting studies using conditioned medium from transfected COS-7 cells suggest that RISC is a secreted protein. Tissue Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues analyzed; high level RISC expression was also observed in human kidney. In situ hybridization verified the localization of RISC to medial SMC of the adult rat aorta. Interestingly, expression in kidney was restricted to proximal convoluted tubules; little or no expression was observed in glomerular cells, distal convoluted and collecting tubules, or medullary cells. Radiation hybrid mapping studies placed the rat RISC locus on chromosome 10q. These studies reveal a novel retinoid-inducible protease whose activity may be involved in vascular wall and kidney homeostasis.

2005; Westbrook et al., 2008). However, the function and significance of this differential regulation of REST during nervous system development and whether the normal low level of REST present in NS/P cells has any roles in maintaining NS/P cell self-renewal and/or differentiation along the different neural lineages remains largely unknown. SUMMARYREST is a master repressor of neuronal genes; however, whether it has any role during nervous system development remains largely unknown. Here, we analyzed systematically the role of REST in embryonic stem cells and multipotent neural stem/progenitor (NS/P) cells, including neurogenic and gliogenic NS/P cells derived from embryonic stem (ES) cells or developing mouse embryos. We showed that REST-null ES cells remained pluripotent and generated teratomas consisting of the three germ layers. By contrast, multipotent NS/P cells lacking REST displayed significantly reduced self-renewal capacity owing to reduced cell cycle kinetics and precocious neuronal differentiation. Importantly, although early-born neurogenic NS/P cells that lack REST were capable of differentiating to neurons and glia, the neuronal and oligodendrocytic pools were significantly enlarged and the astrocytic pool was shrunken. However, gliogenic NS/P cells lacking REST were able to generate a normal astrocytic pool size, suggesting that the shrinkage of the astrocytic pool generated from neurogenic NS/P cells lacking REST probably occurs by default. Microarray profiling of early-born NS/P cells lacking REST showed upregulation of neuronal as well as oligodendrocytic genes, specifically those involved in myelination. Furthermore, chromatin immunoprecipitation analyses showed that some of the upregulated oligodendrocytic genes contain an RE1 motif and are direct REST targets. Together, our data support a central role for REST during neural development in promoting NS/P cell self-renewal while restricting the generation and maturation of neurons and oligodendrocytes. KEY WORDS: Embryonic stem cells, Neural development, Neural stem/progenitor cells, Neurons, Oligodendrocytes, REST, MouseREST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells