Other transcription factor binding sites (
cis-acting elements) are generally located upstream of the core promoter and act through binding of different transcriptional activators and/or repressors to alter the level of gene transcription. In addition to putative binding sites for the following transcription factors such as nuclear factor I (NFI), SP1, Oct-1, C/EBPγ,
etc., the cloned rat LO promoter also contains the hypoxia response element (HRE), the metal response element (MRE) and antioxidant response element (ARE),
etc. [
59]. The cloned rat LO promoter −804/−1 yielding the maximal promoter activity contains three putative NFI-binding sites [
59]. NFI binds to the consensus sequence TTGGC(N5)GCCAA (N = any nucleotides) on duplex DNA as a dimer. Notably, it can also bind to the individual half site, TTGGC or GCCAA, with a somewhat reduced affinity [
62]. The highly conserved N-terminal residues of NFI contain the DNA binding domain whereas the proline-rich C-terminal residues constitute the transcriptional regulation domain [
62]. Four cysteine residues are conserved in the DNA binding domain of all rat NFI isoforms sensitive to oxidative damage [
63,
64]. In addition, the rat LO promoter region −804/−1 also contains four putative hypoxia response elements (HRE, core sequence = RCGTG, R = purine) [
65,
66] including one on the coding strand and three on the noncoding strand at the region −457/−453, −387/−383, −194/−190 and −37/−33 (relative to ATG) [
59], two putative metal response elements (MRE, core sequence = TGCRCNC, R = purine, N = any nucleotide) [
67–
69] located at −269/−263 and −248/−241, and one antioxidant response element (ARE, core sequence = RTGACNNNGC, R = purine, N = any nucleotides) [
70] at the region −581/−572 [
59]. The HIF1 is composed of HIF1α and HIF1β subunits [
71]. The HIF1β is constitutively expressed whereas HIF1α is maintained at a low level in normoxic cells. Upon hypoxia, HIF1α is upregulated and HIF1 complexes bind to HREs transactivating hypoxia-inducible genes [
65]. HIF1α has a unique domain sensitive to oxygen-dependant degradation. In normaxia, specific degradation of HIF1α is triggered through this domain by the proteasome [
66]. Hypoxia is an important complication associated with lung diseases and tumors [
72]. Reduced expression of HIF1α has been detected in the emphysema lung tissues in severe COPD patients [
73], suggesting deregulation of HRE activities occurred in HIF1α targeting genes such as LO under this condition. Since cobalt chloride (CoCl
2) stabilizes the HIF1α protein, this metal ion is used to mimic effects of hypoxia in cell study systems [
74,
75]. The MRE was initially found in multiple copies in metallothionein (MT) genes. MTs are cysteine-rich metal binding proteins essential for cellular metal metabolism and detoxification [
68,
69]. A protein that binds specifically to MREs is termed as MRE-binding transcription factor-1 (MTF-1) [
67]. MTF-1 is a Zn finger transcription factor mediating the expression of MT genes which contain more copies of MREs. Cd and oxidative stress (such as H
2O
2) activate MTF-1 binding to the MT-I gene promoter by binding to or oxidation of other cellular protein sulfhydryls releasing bound Zn that in turn enhances MTF-1 affinity for DNA, thus activating MRE containing genes [
67]. The transcriptional factor interacted with the ARE is the NF-E2-related factor-2 (Nrf2) which drives expressions of a variety xenobiotic metabolizing enzymes such as GST, NQO1, UGT, GCL, HO-1, GSH synthetase, γ-glutamyl transpeptidase,
etc., for antioxidant-detoxification [
70]. Nrf2 is inactive in the cytoplasm by binding to the cysteine-rich Keap1 protein in the actin filaments. Upon oxidative stress, modification of cysteine residues in the Keap1 protein induces the Nrf2 release and nuclear translocation. After forming the heterodimer with Maf, Nrf2 binds to the ARE for transcriptional activation of the genes. CS exposure has been shown to generate reactive oxygen species (ROS) [
16]. Transcription factors for the NFI binding site, HRE, MRE and ARE, are known sensitive to oxidative stress [
62,
66,
69,
70]. These redox-sensitive transcription factors will be addressed as potential targets for CS insult. A schematic linear map of the cloned rat LO promoter is shown in
Figure 2.