Opiorphin-dependent upregulation of CD73 (a key enzyme in the adenosine signaling pathway) in corporal smooth muscle cells exposed to hypoxic conditions and in corporal tissue in pre-priapic sickle cell mice.

The precise molecular mechanisms underlying priapism associated with sickle cell disease remain to be defined. However, there is increasing evidence that upregulated activity of the opiorphin and adenosine pathways in corporal tissue, resulting in heighted relaxation of smooth muscle, have an important role in development of priapism. A key enzyme in the adenosine pathway is CD73, an ecto-5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5) which catalyzes the conversion of adenosine mononucleotides to adenosine. In the present study we investigated how sickle cell disease and hypoxia regulate the interplay between opiorphin and CD73. In the corpora of sickle cell mice we observed significantly elevated expression of both the mouse opiorphin homolog mSmr3a (14-fold) and CD73 (2.2-fold) relative to non-sickle cell controls at a life stage before the exhibition of priapism. Sickle cell disease has a pronounced hypoxic component, therefore we determined if CD73 was also modulated in in vitro corporal smooth muscle (CSM) models of hypoxia. Hypoxia significantly increased CD73 protein and mRNA expression by 1.5-fold and 2-fold, respectively. We previously demonstrated that expression of another component of the adenosine signaling pathway, the adensosine 2B receptor, can be regulated by sialorphin (the rat opiorphin homolologue), and we demonstrate that sialorphin also regulates CD73 expression in a dose- and time-dependent fashion. Using siRNA to knockdown sialorphin mRNA expression in CSM cells in vitro, we demonstrate that the hypoxic upregulation of CD73 is dependent on the upregulation of sialorphin. Overall, our data provide further evidence to support a role for opiorphin in CSM in regulating the cellular response to hypoxia or sickle cell disease by activating smooth muscle relaxant pathways.

Experimental priapism is associated with increased oxidative stress and activation of protein degradation pathways in corporal tissue.

Priapism is a debilitating disease for which there is at present no clinically accepted pharmacological intervention. It has been estimated that priapism lasting more than 24 h in patients is associated with a 44-90% rate of ED. In this investigation, we determined in two animal models of priapism (opiorphin-induced priapism in the rat and priapism in a mouse model of sickle cell disease) if there is evidence for an increase in markers of oxidative stress in corporal tissue. In both animal models, we demonstrate that priapism results in increased levels of lipid peroxidation, glutathione S-transferase activity and oxidatively damaged proteins in corporal tissue. Using western blot analysis, we demonstrated there is upregulation of the ubiquitination ligase proteins, Nedd-4 and Mdm-2, and the lysosomal autophage protein, LC3. The antiapoptotic protein, Bcl-2, was also upregulated. Overall, we demonstrate that priapism is associated with increased oxidative stress in corporal tissue and the activation of protein degradation pathways. As oxidative stress is known to mediate the development of ED resulting from several etiologies (for example, ED resulting from diabetes and aging), we suggest that damage to erectile tissue resulting from priapism might be prevented by treatments targeting oxidative stress.

c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function.

Chromatin organization plays a key role in the regulation of gene expression. The evolutionarily conserved SWI/SNF complex is one of several multiprotein complexes that activate transcription by remodelling chromatin in an ATP-dependent manner. SWI2/SNF2 is an ATPase whose homologues, BRG1 and hBRM, mediate cell-cycle arrest; the SNF5 homologue, INI1/hSNF5, appears to be a tumour suppressor. A search for INI1-interacting proteins using the two-hybrid system led to the isolation of c-MYC, a transactivator. The c-MYC-INI1 interaction was observed both in vitro and in vivo. The c-MYC basic helix-loop-helix (bHLH) and leucine zipper (Zip) domains and the INI1 repeat 1 (Rpt1) region were required for this interaction. c-MYC-mediated transactivation was inhibited by a deletion fragment of INI1 and the ATPase mutant of BRG1/hSNF2 in a dominant-negative manner contingent upon the presence of the c-MYC bHLH-Zip domain. Our results suggest that the SWI/SNF complex is necessary for c-MYC-mediated transactivation and that the c-MYC-INI1 interaction helps recruit the complex.

High-resolution mapping of the X-linked hypohidrotic ectodermal dysplasia (EDA) locus.

The X-linked hypohidrotic ectodermal dysplasia (EDA) locus has been previously localized to the subchromosomal region Xq11-q21.1. We have extended our previous linkage studies and analyzed linkage between the EDA locus and 10 marker loci, including five new loci, in 41 families. Four of the marker loci showed no recombination with the EDA locus, and six other loci were also linked to the EDA locus with recombination fractions of .009-.075. Multipoint analyses gave support to the placement of the PGK1P1 locus proximal to the EDA locus and the DXS453 and PGK1 loci distal to EDA. Further ordering of the loci could be inferred from a human/rodent somatic cell hybrid derived from an affected female with EDA and an X;9 translocation and from studies of an affected male with EDA and a submicroscopic deletion. Three of the proximal marker loci, which showed no recombination with the EDA locus, when used in combination, were informative in 92% of females. The closely linked flanking polymorphic loci DXS339 and DXS453 had heterozygosities of 72% and 76%, respectively, and when used jointly, they were doubly informative in 52% of females. The human DXS732 locus was defined by a conserved mouse probe pcos169E/4 (DXCrc169 locus) that cosegregates with the mouse tabby (Ta) locus, a potential homologue to the EDA locus. The absence of recombination between EDA and the DXS732 locus lends support to the hypothesis that the DXCrc169 locus in the mouse and the DXS732 locus in humans may contain candidate sequences for the Ta and EDA genes, respectively.