Anthony P. Young

Therapeutic implications of human endothelial nitric oxide synthase gene polymorphism

Vascular endothelial dysfunction is now recognized as a common phenomenon in an array of cardiovascular disorders. Production of nitric oxide via the endothelial isoform of nitric oxide synthase [eNOS (previously termed NOS3 or ecNOS)] is vital for a healthy endothelium; several polymorphic variations of the gene encoding eNOS (NOS3) are now known and have been investigated with respect to disease risk. Surprisingly, only approximately half of these studies have demonstrated significant associations between NOS3 polymorphisms and cardiovascular disease, and many reports are contradictory. Central issues include adequate statistical power, appropriateness of control cohorts, multigene interactions and plausible biological consequences. So far, the inconsistencies are not unique to the NOS3 polymorphisms, but probably represent the broad challenges in defining genetic aspects of complex disease processes.

Differential Regulation of the Two Neuronal Nitric-oxide Synthase Gene Promoters by the Oct-2 Transcription Factor

The Oct-2 transcription factor has been shown previously to repress both the cellular tyrosine hydroxylase and the herpes simplex virus immediate-early genes in neuronal cells. Here we identify the gene encoding the neuronal nitric-oxide synthase (nNOS) as the first example of a gene activated in neuronal cells by Oct-2. The levels of the nNOS mRNA and protein are greatly reduced in neuronal cell lines in which Oct-2 levels have been reduced by an antisense method, although these cells have enhanced levels of tyrosine hydroxylase. Moreover, the nNOS gene regulatory region is activated by Oct-2 expression vectors upon cotransfection into both neuronal and non-neuronal cells, and this response is dependent upon a 20-amino acid region within the COOH-terminal activation domain of Oct-2. Of the two closely linked promoters that drive nNOS gene expression, only the downstream 5.1 promoter is activated by Oct-2, whereas the 5.2 promoter is unaffected. These effects are discussed in terms of the potential role of Oct-2 in regulating nNOS expression in the nervous system.

A promoter element with enhancer properties, and the orphan nuclear receptor RORα, are required for Purkinje cell-specific expression of a Gi/o modulator

The promoter and structural portion of the gene, Pcp-2(L7), has frequently been used to target expression of proteins to cerebellar Purkinje cells. In our continuing analysis of the transcription of this gene and how it relates to the G-protein and Ca2+ channel modulatory functions of the encoded protein, we have dissociated the promoter and structural gene and identified cooperative functions. A 0.9 kb fragment of the proximal promoter has positional properties of a classical enhancer, yet its function requires the presence of the structural gene. We demonstrate that RORalpha, the gene product of the mutant mouse locus called staggerer (Rora(sg)), binds to and activates expression through this promoter element using functional assays in vitro and in vivo. The structural gene has a repressive effect on gene expression outside Purkinje cells, and likely participates in the suppression of Pcp-2(L7) gene expression in the many other brain and non-neuronal cell types, besides Purkinje cells, known to express RORalpha. Additional studies in vivo show that while Pcp-2(L7) expression is dependent on RORalpha throughout the cerebellum, this dependence is greatest in the intermediate region between the vermis and far lateral hemispheres. Thus, in addition to its recently indicated role in Ca2+-mediated reciprocal cell-cell signaling in Purkinje cells, RORalpha may also contribute to functional differences in cerebellar subregions.

A transcriptional enhancer of the glutamine synthetase gene that is selective for retinal Müller glial cells.

This article demonstrates that the chicken glutamine synthetase (GS) promoter contains cis-acting elements that direct transcription to retinal Müller glial cells. The transient assay system developed to identify these elements involved electroporation of intact retinal tissue with GS-β-galactosidase fusion genes followed by preparation of primary cultures and histochemical assay of cells expressing β-galactosidase. Plasmids containing β-galactosidase under transcriptional control by two different viral promoters are expressed primarily in neuronal cells after transfection of intact embryonic d 12 retina. In sharp contrast, expression is primarily in Müller glia after transfection with a GS-β-galactosidase fusion gene. Although GS is glucocorticoid inducible, steroid hormone is not required to achieve Müller cell-selective expression of the GS-β-galactosidase fusion gene. Deletion studies indicate that multiple cis-acting elements located between nucleotides −436 and −61 relative to the GS transcription start site contribute to produce Müller cell selectivity. Moreover, these upstream elements enhance expression of a heterologous promoter in Müller cells but not neurons. These results indicate that an enhancer located between 61 and 436 nucleotides upstream of the transcription start site contributes to Müller cell-selective expression of the GS gene in the retina.

Transcription of the Human Neuronal Nitric Oxide Synthase Gene in the Central Nervous System is Mediated by Multiple Promoters

Publisher Summary The principal contribution described in this chapter is the identification and initial characterization of two distinct promoters for human NOS1 gene transcription. Although closely linked, these promoters are separable, in that they can independently drive the expression of appropriately linked reporter genes in transfection systems. The demonstration of independent promoter activities renders it unlikely that alternative splicing of a single primary transcript produces the heterogeneity in NOS1 mRNA structure reflected in the cDNA clones pNOS5′1 and pNOS5′2. The number of NOS1 expressing neurons and the level of NOS1 mRNA rise sharply then decline during development of the chicken tectum. Recent pharmacological studies indicate that induction of NOS1 might be crucial to establishing the appropriate pattern of synaptic connections in the tectum. Moreover induction of NOS1 in the tectum appears to require projection of axons from retinal ganglion cells. Potential mechanisms for these alterations in NOS1 gene expression during CNS development is anticipated to emerge through studies of NOS1 promoter function. It is also important to characterize the genetic basis for NOS1 gene expression in the CNS because the NOS1 promoter is apt to provide a valuable research tool. NOS+ neurons are selectively resistant to degeneration in Huntingtons chorea and Alzheimers disease, during ischemia, and in response to assaults by the neurotoxin quinolinic acid. In addition, NOS1 is dramatically induced in spinal motor neurons as well as in dorsal root ganglia, in response to avulsion and proximal transection, respectively.

Steroid Hormone Receptors Activate Transcription in Glial Cells of Intact Retina but not in Primary Cultures of Retinal Glial Cells

We have compared the steroid responsiveness of Müller glial cells of intact embryonic chicken retina with that of primary cultures derived from Müller glia. Appropriately constructed fusion genes were found to be highly glucocorticoid inducible after their cotransfection with an expression vector encoding the human glucocorticoid receptor (GR) into intact embryonic d 10 (E10) or E5.5 retina. Dramatically attenuated inductions were obtained after cotransfection of Müller cell primary cultures. The progesterone receptor (PR) was also demonstrated to function in intact retina, but not in Müller cell primary cultures. An immunochemical assay was utilized to confirm that a glucocorticoid-responsive, β-galactosidase-encoding fusion gene was specifically induced in Müller cells after its transfection into intact retina. Thus, in contrast to Müller cells in intact retina, Müller cells in primary culture have lost the capacity to achieve transcriptional activation by steroid receptors. We postulate that coordinate expression of the GR, and other more general factors required for steroid inducibility, is lost by dispersion and primary culture of retinal Müller glial cells.