Promoter Expression and Regulation by Regulatory DNA Elements and Transcription Factors

The primary site of channel expression control is in the expression and function of the promoter of the genes encoding the channel proteins. Silencers are ds-acting regulatory DNA elements that downregulate gene transcription in a cell/tissue-specific fashion in order to either control cell-specific gene expression or prevent the negative consequences of overexpression. DNA elements that are important in the transcriptional regulation of Kv1.5, Kv1.4, and Kv3.1 genes have been identified (104). In the case of the cardiac Kv1.5 channel, a Kv1.5 represser element (KRE) located in the 5' flanking region of the Kv1.5 gene, contains a dinucleotide repetitive element that is necessary for mediating silencer activity (58, 104). KRE selectively decreases the expression of Kv1.5 in cell lines that do not express Kv1.5 proteins. Deletion of the KRE repetitive element abolishes the silencing activity in transfected cells. In addition, a KRE binding factor (KBF) has recently been identified, which may regulate the cell-specific expression of Kv1.5 by abolishing the silencer effects of KRE. Whether the KRE or KBF is involved in regulating Kv1.5 transcription and expression in PASMC during hypoxia has not been evaluated. However, we can speculate that the expression of the KRE in the Kvl.5 gene (and in other Kv channel a subunit genes) may play an important role in the hypoxia-induced downregulation of Kv channel expression in PASMC. Alternatively, hypoxia may also exert its inhibitory effect on Kv channel a subunit expression by downregulating KBF expression, thereby allowing for silencing of Kv1.5 (and other Kv channels) gene expression and decreasing functional Kv channel availability.

It is generally accepted that changes in P02 can regulate gene expression via second messengers, protein kinases, and transcription factors such as activating protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), nuclear factor-KB (NF-kB), nuclear factor interleukin-6 (NF-IL6), and early growth response-1 (EGR-1) (92). Transcription factors modulate channel protein expression by regulating genes that contain binding sites within their promoters (55). Two AP-1 family members, c-Fos and c-Jun, have been suggested as possible effectors of hypoxic gene regulation based on their ability to sense changes in redox potential and levels of cytoplasmic and nuclear [Ca2+], thereby modulating transcription and expression of the AP-1-responsive genes (1, 6). Under normoxic conditions, overexpression of c-jun in rat PASMC resulted in decreased Kvl.5 channel expression and activity, as well as increased Kv channel P subunit expression and increased current inactivation (113). The subsequent depolarization and Ca2+ increase enhanced cell proliferation. The divergent regulatory effects of c-Jun on Kv channel a (e.g., Kvl.5) and P (e.g., Kvp2) subunit gene expression may be due to direct binding to the AP-1 binding sites of the channel gene promoter (as has been reported for the human MaxiK gene, hslo ) (20) or to inducing expression of an intermediate that subsequently inhibits the channel expression.

We do have evidence that AP-1 binding activity is increased during chronic hypoxia (3% 02, 72 hrs) in human pulmonary artery endothelial cells (25). Whether the AP-1 binding activity is regulated by chronic hypoxia in PASMC and whether the hypoxia-sensitive Kv channel genes all contain the AP-1 binding sites in their promoters remain unresolved.

While AP-1 binding may be involved more in cellular proliferation and the pulmonary remodeling induced by prolonged hypoxia, HIF-1 activation is intimately involved in hypoxia-induced pulmonary hypertension (111). The biological activity of HIF-1 is regulated by the expression and activity of the HIF-la subunit. Under normoxic conditions, HIF-1 is ubiquitinated and rapidly degraded, thereby never allowed to form functional heterodimeric HIF-1 proteins. Under hypoxic conditions, HIF-i a ubiquitination is inhibited and HIF-la dimerizes with HIF-1 P subunits to form functional HIF-1 which is responsible for activation of a number of target genes including vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1). The role of these growth factors and mitogens in HPV is discussed in the following section.

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