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Prostate tumorigenesis is associated with loss of PTEN gene expression. We and others have recently reported that PTEN is regulated by Notch-1 signaling. Herein, we tested the hypothesis that alterations of the Notch-1 signaling pathway are present in human prostate adenocarcinoma and that Notch-1 signaling regulates PTEN gene expression in prostate cells. Prostate adenocarcinoma cases were examined by immunohistochemistry for ligand cleaved (activated) Notch-1 protein. Tumor foci exhibited little cleaved Notch-1 protein, but expression was observed in benign tissue. Both tumor and benign tissue expressed total (uncleaved) Notch-1. Reduced Hey-1 expression was seen in tumor foci but not in benign tissue, confirming loss of Notch-1 signaling in prostate adenocarcinoma. Retroviral expression of constitutively active Notch-1 in human prostate tumor cell lines resulted in increased PTEN gene expression. Incubation of prostate cell lines with the Notch-1 ligand, Delta, resulted in increased PTEN expression indicating that endogenous Notch-1 regulates PTEN gene expression. Chromatin immunoprecipitation demonstrated that CBF-1 was bound to the PTEN promoter. These data collectively indicate that defects in Notch-1 signaling may play a role in human prostate tumor formation in part via a mechanism that involves regulation of the PTEN tumor suppressor gene.

The human GH (hGH) gene cluster is regulated by a remote 5 locus control region (LCR). HSI, an LCR component located 14.5 kb 5 to the hGH-N promoter, constitutes the primary determinant of highlevel hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast, the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary.T he expression of the GH gene has been the focus of intense study over the past 20 years. A consistent feature of all GH promoters is a highly conserved pair of AT-rich binding sites for the pituitary-restricted trans factor Pit-1 (1, 2). Between these two Pit-1 elements is a conserved binding site for a ubiquitous DNA-binding protein, Zn15 (3). Additional, less conserved cis elements have been identified in the vicinity of the GH promoter in various species. Studies carried out in vitro and in celltransfection models indicate that the Pit-1 binding sites are essential to the expression of the GH gene (4-6). However, studies of human (h)GH gene expression in transgenic settings demonstrate that promoter-proximal elements including the Pit-1 sites are not sufficient to activate hGH gene expression in vivo. For example, in transgenic mice, the hGH gene with its full promoter and as much as 7.5 kb of 5Ј-flanking sequences is either not expressed or expressed at very low levels and does not maintain somatotrope cell specificity (7,8). These data suggest that regulatory sequences quite distant from the hGH gene are necessary to establish a transcriptionally active chromatin domain.The hGH gene cluster spans 48 kb on chromosome 17q22-24 (for details see Fig. 1; ref. 9). This cluster contains the pituitaryexpressed hGH-N and four genes expressed in the placenta:, and hCS-B (Fig. 1). A search for distal regulatory determinants of hGH-N gene expression revealed DNaseI hypersensitive sites (HSs) in pituitary chromatin and a partially overlapping set of HSs in placental chromatin (ref. 8; Fig. 1). HSI and HSII, located between Ϫ14.5 kb and Ϫ15 kb relative to the hGH-N promoter, are specific to pituitary chromatin. HSIV, at Ϫ30 kb, is specific to the placenta and HSIII and HSV, at Ϫ27.5 and Ϫ32 kb, respectively, are shared in both expressing tissues. The five HSs together comprise a putative hGH locus control region (LCR). Significantly, when hGH-N transgene DNA is extended to encompass the entire set of HSs, hGH gene expression is rendered pituitary-specific, copy number-dependent, and robust in every line generat...

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP.S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/ 239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/ WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP.S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.