Eukaryotic gene transcription requires the coordinated assembly of upstream cis-element binding proteins, intermediary cofactors, and components of the basal transcription machinery into a multicomponent complex competent to initiate transcription. During this process, sequence-specific DNA-binding transcriptional activators and/or repressors play a pivotal role in modulating the cell-type specific expression of genes. While most such proteins bind to double-stranded DNA target sequences, a small but intriguing subclass has been identified that show enhanced affinity and specificity for either the sense or antisense strands of certain cis-regulatory elements required for promoter-specific activation (1-4) or repression (5-9). We have recently cloned and identified two single-stranded DNA (ssDNA)

Smooth muscle cells (SMC) of the vascular wall, bladder, myometrium, and gastrointestinal and respiratory tracts retain the ability to proliferate postnatally, which enables adaptive responses to injury, hormonal, or mechanical stimulation. SMC growth is regulated by a number of mesenchymal growth factors, including insulin-like growth factor I (IGF-I). To explore the function of IGF-I on SMC in vivo, the mouse SMC ␣ -actin promoter fragment SMP8 ( Ϫ 1074 bp, 63 bp of 5 Ј UT and 2.5 kb of intron 1) was cloned upstream of rat IGF-I cDNA, and the fusion gene microinjected to fertilized eggs of the FVB-N mouse strain. Mating of hemizygous mice with controls produced about 50% transgenic offspring, with equal sex distribution. Transgenic IGF-I mRNA expression was confined to SMC-containing tissues, with the following hierarchy: bladder Ͼ stomach Ͼ aorta ϭ uterus Ͼ intestine. There was no transgene expression in skeletal muscle, heart, or liver. Radioimmunoassayable IGF-I content was increased by 3.5-to 4-fold in aorta, and by almost 10-fold in bladder of transgenic mice at 5 and 10 wk, with no change in plasma IGF-I levels. Wet weight of bladder, stomach, intestine, uterus, and aorta was selectively increased, with no change in total body or carcass weight of transgenic animals. In situ hybridization showed that transgene expression was exquisitely targeted to the smooth muscle layers of the arteries, veins, bladder, ureter, stomach, intestine, and uterus. Paracrine overproduction of IGF-I resulted in hyperplasia of the muscular layers of these tissues, manifesting in remarkably different phenotypes in the various SMC beds. Whereas the muscular layer of the bladder and stomach exhibited a concentric thickening, the SMC of the intestine and uterus grew in a longitudinal fashion, resulting in a marked lengthening of the small bowel and of the uterine horns. This report describes the first successful targeting of expression of any functional protein capable of modifying the phenotype of SMC in transgenic mice. IGF-I stimulates SMC hyperplasia, leading to distinct patterns of organ remodeling in the different tissue environments. (

Abstract-In mammalian organs under normoxic conditions, O 2 concentration ranges from 12% to Ͻ0.5%, with O 2 Ϸ14%in arterial blood and Ͻ10% in the myocardium. During mild hypoxia, myocardial O 2 drops to Ϸ1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO 2 results in perceived hyperoxia. These effects were independent of NADPH oxidase function. CFs exposed to high O 2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-␤1, (2) lowered telomerase activity, and (3) Key Words: redox Ⅲ free radicals Ⅲ heart Ⅲ cell culture C ellular O 2 concentrations are maintained within a narrow range (normoxia) because of the risk of oxidative damage from excess O 2 (hyperoxia) and of metabolic demise from insufficient O 2 (hypoxia). 1 PO 2 ranges from 90 to Ͻ3 mm Hg in mammalian organs under normoxic conditions, with arterial PO 2 of Ϸ100 mm Hg or Ϸ14% O 2 . 2 Thus, "normoxia" for cells is a variable that is dependent on the specific localization of the cell in organs and functional status of the specific tissue. O 2 sensing is required to adjust to physiological or pathophysiological variations in PO 2 . Current work in this field is almost exclusively focused on the study of hypoxia. Reoxygenation, on the other hand, has been mostly investigated in the context of oxidative injury. Over 25 years ago, it was observed that PO 2 beyond the comfort of the "perceived normoxic range" is a significant stressor, leading to growth arrest. 3 The molecular bases of such observations remain to be characterized in light of current knowledge of signal transduction.During chronic hypoxia in the heart, cells adjust their normoxic set point such that the return to normoxic PO 2 after chronic hypoxia is perceived as relative hyperoxia. 4,5 We hypothesized that such challenge triggers changes in signal transduction processes. Although acute insult caused during reperfusion may be lethal to cells localized at the focus of insult, elevation of O 2 tension in the surrounding ischemic tissue triggers phenotypic changes in the surviving cells that may be associated with tissue remodeling.Ischemia in the heart results in a hypoxic area containing a central focus of near-zero O 2 pressure bordered by tissue with diminished but nonzero O 2 pressures. These border zones extend for several millimeters from the hypoxic core, with the O 2 pressures progressively increasing from the focus to the normoxic region. 6 Moderate hypoxia is associated with a 30% to 60% decrease (Ϸ1% to 3% O 2 ) in PO 2 . 7 Cardiac fibroblasts (CFs) are mainly responsible for the synthesis of major extracellular matrix (ECM) in the myocardium, including fibrillar collagen types I and III and fibronectin. More than 90% of the interstitial cells of the myocardium are fibroblasts, 8 which actively e...

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor ␤1 (TGF-␤1) and accompanied by transient activation of the vascular smooth muscle ␣-actin (Sm␣A) gene. Multiple positive-regulatory elements were identified as essential mediators of basal Sm␣A enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-␤1. However, TGF-␤1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the Sm␣A enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate Sm␣A enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the Sm␣A enhancer required for TGF-␤1 amplification. Additionally, the TGF-␤1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for Sm␣A enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the Sm␣A enhancer. The identification of Sp and Smad proteins as essential, independent activators of the Sm␣A enhancer provides new insight into the poorly understood process of myofibroblast differentiation.

The mouse vascular smooth muscle alpha-actin (SMA) gene enhancer is activated in fibroblasts by transforming growth factor beta1 (TGFbeta1), a potent mediator of myofibroblast differentiation and wound healing. The SMA enhancer contains tandem sites for the Sp1 transcriptional activator protein and Puralpha and beta repressor proteins. We have examined dynamic interplay between these divergent proteins to identify checkpoints for possible control of myofibroblast differentiation during chronic inflammatory disease. A novel element in the SMA enhancer named SPUR was responsible for both basal and TGFbeta1-dependent transcriptional activation in fibroblasts and capable of binding Sp1 and Pur proteins. A novel Sp1:Pur:SPUR complex was dissociated when SMA enhancer activity was increased by TGFbeta1 or Smad protein overexpression. Physical association of Pur proteins with Smad2/3 was observed as was binding of Smads to an upstream enhancer region that undergoes DNA duplex unwinding in TGFbeta1-activated myofibroblasts. Purbeta repression of the SMA enhancer could not be relieved by TGFbeta1, whereas repression mediated by Puralpha was partially rescued by TGFbeta1 or overexpression of Smad proteins. Interplay between Pur repressor isoforms and Sp1 and Smad coactivators may regulate SMA enhancer output in TGFbeta1-activated myofibroblasts during episodes of wound repair and tissue remodeling.