Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-beta) and the liphophilic hormone vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways.

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identi®ed a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor a (hERa) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hERa A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogenbound hERa in MCF7 cells and in partially puri®ed complexes associated with hERa from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hERa and TIF2 in the nucleus. The presence of p72/ p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hERa. These ®nd-ings indicate that p72/p68 acts as an ER subtypeselective coactivator through ERa AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.

Cited2 is a cAMP-responsive element-binding protein (CBP)͞p300 interacting transcriptional modulator and a proposed negative regulator for hypoxia-inducible factor (HIF)-1␣ through its competitive binding with HIF-1␣ to CBP͞p300. Disruption of the gene encoding Cited2 is embryonic lethal because of defects in the development of heart and neural tube. Morphological and Doppler echocardiographic analyses of Cited2 ؊/؊ embryos reveal severe cardiovascular abnormalities, including pulmonic arterial stenosis and ventricular septal defects accompanied by high peak outflow velocities, features of the human congenital cardiac defect termed tetralogy of Fallot. The mRNA levels of several HIF-1␣-responsive genes, such as vascular endothelial growth factor (VEGF), Glut1, and phosphoglycerate kinase 1, increased in the Cited2 ؊/؊ hearts. The increase of VEGF levels is significant, because defects in the Cited2 ؊/؊ embryos closely resemble the major defects observed in the VEGF transgenic embryos. Finally, compared with wild-type, cultured fibroblasts from Cited2 ؊/؊ embryos demonstrate an enhanced expression of HIF-1␣-responsive genes under hypoxic conditions. These observations suggest that functional loss of Cited2 is responsible for defects in heart and neural tube development, in part because of the modulation of HIF-1 transcriptional activities in the absence of Cited2. These findings demonstrate that Cited2 is an indispensable regulatory gene during prenatal development.

Disrupted ERK1/2 (MAPK3/MAPK1) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel Ϸ1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2. These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade (B-Raf and C-Raf, MEK1and MEK2) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/ serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.22q11 microdeletion ͉ human syndromes ͉ MAP kinase

Light has been used to noninvasively alter the excitability of both neural and cardiac tissue 1–10. Recently, pulsed laser light has been shown to be capable of eliciting action potentials in peripheral nerves and in cultured cardiomyocytes 7–10. Here, we demonstrate for the first time optical pacing (OP) of an intact heart in vivo. Pulsed 1.875 μm infrared laser light was employed to lock the heart rate to the pulse frequency of the laser. A laser Doppler velocimetry (LDV) signal was used to verify the pacing. At low radiant exposures, embryonic quail hearts were reliably paced in vivo without detectable damage to the tissue, indicating that OP has great potential as a tool to study embryonic cardiac dynamics and development. In particular, OP can be utilized to control the heart rate, and thereby alter stresses and mechanically transduced signaling.

A phage endoneuraminidase that specifically cleaves alpha-2, 8-1inked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.The neural cell adhesion molecule (NCAM) t is a cell-surface glycoprotein that serves as a ligand in the formation of cellcell bonds (see references 9, 26, and 27 for review). The binding appears to involve the direct interaction between NCAMs on each cell, and therefore represents an example of homophilic adhesion (31). NCAM is expressed on a variety of cell types in the vertebrate embryo, including primitive neuroepithelia (36), neurons (29, 32), glial cells (14, 23), and muscle cells (15). Adhesion of neurons to muscle, glia, and other neurons has been found to be mediated at least in part by NCAM, and appears to be an essential event in the formation of nerve-muscle contacts that lead to synapses ~ Abbreviations used in this paper: endo-N, the soluble endoneuraminidase from K1F bacteriophage; NCAM, neural cell adhesion molecule.(30), guidance of axon growth cones along marginal pathways of the central nervous system (36), and the formation of neurite fascicles (29). In addition, antibodies to NCAM have been shown to alter the histogenesis of the retina in vitro (3).The complex glycan chains associated with NCAM have a very unusual structure including one or more relatively long unbranched homopolymers of alpha-2, 8-1inked sialic acid residues (6,7,12,43). Removal of the sialic acid by commercial exoneuraminidases results in an increase in the app...

The embryonic avian heart is an important model for studying cardiac developmental biology. The mechanisms that govern the development of a four-chambered heart from a peristaltic heart tube are largely unknown due in part to a lack of adequate imaging technology. Due to the small size and rapid motion of the living embryonic avian heart, an imaging system with high spatial and temporal resolution is required to study these models. Here, an optical coherence tomography (OCT) system using a buffered Fourier Domain Mode Locked (FDML) laser is applied for ultrahigh-speed non-invasive imaging of embryonic quail hearts at 100,000 axial scans per second. The high scan rate enables the acquisition of high temporal resolution 2D datasets (195 frames per second or 5.12 ms between frames) and 3D datasets (10 volumes per second). Spatio-temporal details of cardiac motion not resolvable using previous OCT technology are analyzed. Visualization and measurement techniques are developed to non-invasively observe and quantify cardiac motion throughout the brief period of systole (less than 50 msec) and diastole. This marks the first time that the preseptated embryonic avian heart has been imaged in 4D without the aid of gating and the first time it has been viewed in cross section during looping with extremely high temporal resolution, enabling the observation of morphological dynamics of the beating heart during systole.

Current therapies for diseases of heart muscle (cardiomyopathy) and aorta (aortopathy) include inhibitors of the renin-angiotensin system, β-adrenergic antagonists, and the statin class of cholesterol-lowering agents. These therapies have limited efficacy, as adverse cardiovascular events continue to occur with some frequency in patients taking these drugs. Although cardiomyopathy and aortopathy can coexist in a number of conditions (for example, Marfan's syndrome, acromegaly, pregnancy, and aging), pathogenetic molecular links between the two Copyright 2010 by the American Association for the Advancement of Science; all rights reserved. † To whom correspondence should be addressed. mukesh.jain2@case.edu. * These authors contributed equally to this work. SUPPLEMENTARY MATERIALwww.sciencetranslationalmedicine.org/cgi/content/full/2/26/26ra26/DC1 Fig. S1. Cardiovascular abnormalities in AngII-treated mice and cultured cells. Fig. S2. Baseline abnormalities in Klf15 −/− heart and aorta. Fig. S3. Cardiac mass and systolic blood pressure after AngII infusion. Fig. S4. Histologic parameters in aortas. Fig. S5. MMP-3 abundance in aortic smooth muscle. Fig. S6. p53 mRNA concentrations in heart and aortic tissue. Fig. S7. p300 abundance in hearts, curcumin administration protocol, and aortic morphometry after curcumin therapy. Table S1. Baseline cardiac parameters in Klf15 −/− and wild-type mice. Table S2. Cardiac parameters in Klf15 −/− and wild-type mice after AngII infusion. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diseases remain poorly understood. We reasoned that identification of common molecular perturbations in these two tissues could point to therapies for both conditions. Here, we show that deficiency of the transcriptional regulator Kruppel-like factor 15 (Klf15) in mice leads to both heart failure and aortic aneurysm formation through a shared molecular mechanism. Klf15 concentrations are markedly reduced in failing human hearts and in human aortic aneurysm tissues. Mice deficient in Klf15 develop heart failure and aortic aneurysms in a p53-dependent and p300 acetyltransferase-dependent fashion. KLF15 activation inhibits p300-mediated acetylation of p53. Conversely, Klf15 deficiency leads to hyperacetylation of p53 in the heart and aorta, a finding that is recapitulated in human tissues. Finally, Klf15-deficient mice are rescued by p53 deletion or p300 inhibition. These findings highlight a molecular perturbation common to the pathobiology of heart failure and aortic aneurysm formation and suggest that manipulation of KLF15 function may be a productive approach to treat these morbid diseases.