C/EBP-β Mediates iNOS Induction by Hypoxia in Rat Pulmonary Microvascular Smooth Muscle Cells
Exposure of rats to 10% O2 for 4 days caused pulmonary hypertension and induced expression of both inducible nitric oxide synthase (iNOS) and CCAAT box enhancer binding protein-β (C/EBP-β) in rat lung. Electrophoretic mobility shift assays (EMSAs) showed that exposure to 1% O2 increased the C/EBP-β binding in rat pulmonary microvascular smooth muscle cells (rPSMs). To test the hypothesis that C/EBP-β participates in hypoxia-induced iNOS expression in rPSMs, a C/EBP motif at −910 bp of rat iNOS promoter was mutated. rPSMs transfected with the rat iNOS promoter and exposed to 1% O2 for 24 hours had significantly increased wild-type iNOS promoter activity. The hypoxia-induced promoter activity was abolished by the C/EBP motif mutation. Thus, C/EBP-β mediates, at least in part, hypoxia-induced iNOS expression in rPSMs.
Inducible nitric oxide synthase (iNOS) is upregulated in rat pulmonary vasculature1,2⇓ and cardiac myocytes3 in response to hypoxia. How iNOS is upregulated by hypoxia is not clear. HIF-1α, a well-established hypoxia-inducible transcription factor,4 was found upregulated in rat pulmonary microvascular endothelial cells (rPMECs) in response to hypoxia; disruption of the HIF binding site in murine iNOS promoter abolished hypoxia-induced murine iNOS promoter activation in rPMECs,5 suggesting that HIF-1α might mediate hypoxia-induced iNOS expression in rPMECs. However, unlike the murine iNOS promoter, there is no fully matched HIF binding site in the currently cloned rat (accession No. AF04208) or human (accession No. AF017634) iNOS promoter, suggesting that factors other than HIF-1α may mediate iNOS induction by hypoxia in rats and humans. Furthermore, HIF-1α activation by hypoxia in rat pulmonary arterial smooth muscle cells (rPASMs) is not universally observed. Some studies report that hypoxia slightly enhanced the HIF-1α activation in rPASMs.6 Others report that hypoxia inhibited the HIF-1α activation in rPASMs.7 In addition, increasing evidence suggests that HIF-1α activation is regulated not only by oxygen, but also by other factors, such as serum8 and nitric oxide.9 Thus, HIF-1α may not be the only transcription factor that mediates iNOS induction by hypoxia.
CAAT box enhancer binding proteins (C/EBPs) consist of a family of transcription factors with structural and functional homology, which currently includes six C/EBPs, designated C/EBP-α, -β, -γ, -δ, -ε, and -ζ.10 C/EBPs recognize and bind to specific sequences, ATTGCGCAAT, a strong binding site, and TKNNGYAAK, a weak binding site, as either homodimers or heterodimers.11 Among C/EBPs, C/EBP-β and C/EBP-δ are strongly upregulated at the transcription level by inflammatory stimuli. C/EBP-β participates in both inflammatory and metabolic gene regulations. It plays a role in both initiation and maintenance of gene expression.12 C/EBP-β participates in iNOS gene regulation. Its binding to a C/EBP motif located at −172 bp of the rat iNOS promoter mediates the cAMP-induced promoter activity in rat mesangial cells.13 Mutation of the corresponding C/EBP motif in the murine iNOS promoter decreases the promoter activity induced by lipopolysaccharide plus interferon-γ in a mouse MTAL cell line14 and a mouse macrophage cell line J774.A1.15
Materials and Methods
For the complete Materials and Methods section, please see the online data supplement available at http://www.circresaha.org.
Results and Discussion
It has been suggested that C/EBP-β is upregulated in hypoxic murine lung.16 To test the hypothesis that C/EBP-β is upregulated in hypoxic rat lung, we exposed 18 male Sprague-Dawley rats (200 to 300 g) to room air (normoxic group, 9 rats) or 10% O2 (hypoxic group, 9 rats) for 4 days. Compared with normoxic rats, the hypoxic group had much higher mean pulmonary arterial pressure (24.3±4.7 versus 14.6±2.3 mm Hg, P<0.01) and a much higher right ventricular weight/left ventricular and septum weight ratio (0.336±0.012 versus 0.240±0.013, P<0.001), a criterion for right ventricular hypertrophy. The results indicate that exposure to 10% O2 for 4 days induced pulmonary hypertension in the rats. Consistent with previous studies,2 Western blots showed that iNOS was upregulated in the hypoxic lung (Figure 1). As shown in Figure 1, the transcription factor C/EBP-β was indeed upregulated in the hypoxic rat lung. This is the first evidence that C/EBP-β is upregulated by hypoxia in rat lung. Double-immunofluorescent staining with smooth muscle α-actin and C/EBP-β showed that the hypoxia-upregulated C/EBP-β was mainly distributed in the smooth muscle of pulmonary artery (Figure 1).
To test whether C/EBP-β is induced by hypoxia in the cultured rat pulmonary microvascular smooth muscle cells (rPSMs), rPSMs were exposed to 1% O2/5% CO2 balanced with N2 or to room air for 24 hours in the presence or absence of IL-1β (100 U/mL). The nuclear protein was then isolated and subjected to electrophoretic mobility shift assay (EMSA), using a C/EBP consensus sequence (5′-TGCAGATTGCGCAATCTGCA-3′) as probe. It was observed that hypoxia alone induced C/EBP-β binding; however, hypoxia had no significant effect on IL-1β–induced C/EBP-β binding (Figure 2A). The results indicate that hypoxia can induce C/EBP-β in rPSMs.
Both the human and rat iNOS promoters contain the TKNNGYAAK binding sites of C/EBP. In previous studies, we identified a functional C/EBP binding motif located at −910 to −902 bp of the rat iNOS promoter that mediated iNOS induction by IL-1β in rat aortic smooth muscle cells.17 In the present study, by cold-probe competition, we demonstrated that C/EBP-β in rPSMs was bound to the C/EBP motif of rat iNOS promoter. The wild-type or mutated rat iNOS promoter fragment (−921 to −898 bp) that contained the C/EBP motif was used as cold probe. The wild-type probe abolished the C/EBP-β binding to the hot probe, but for the C/EBP motif mutated probe, either deletion of −914 to −905 bp, or substitution of GG for AA at −904 to −903 bp, did not (Figure 2B).
To test our hypothesis that the hypoxia-activated C/EBP-β might mediate iNOS upregulation in rPSMs via binding to the C/EBP motif located at −910 bp of rat iNOS promoter, rat iNOS promoter analysis was performed. Two wild-type rat iNOS promoter constructs, −1.4 kb (≈60% promoter activity) and −3.2 kb (full promoter activity),18 were used in the studies. Hypoxia (1% O2) for 24 hours increased the activity of the wild-type promoters in rPSMs (Figure 3). The C/EBP motif deletion from the −1.4 kb promoter abolished the hypoxia-induced promoter activity (Figure 3). This deletion abolishes both the IL-1β–induced and IFN-γ–enhanced promoter activity and may disrupt both the C/EBP motif and the overlapping IRF motif (−907 to −917 bp).17 To exclude the involvement of the overlapping IRF site, the substitution of GG for AA at −904 to −903 bp of the −3.2-kb promoter was generated. The substitution mutation also abolished the hypoxia-induced promoter activity (Figure 3). Thus, C/EBP-β upregulation by hypoxia may mediate, at least in part, the hypoxia-induced iNOS upregulation in rPSMs via binding at the C/EBP motif located at −910 bp of the rat iNOS promoter. These results are the first evidence showing linkage between hypoxia-induced C/EBP-β activation and hypoxia-induced iNOS expression in rat lung.
This study is supported by HL39706 (R.A.J.) from the National Heart, Lung, and Blood Institute and by GM49111 (R.A.J.) from the National Institute of General Medical Sciences.
This manuscript was sent to Elizabeth G. Nabel, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received July 20, 2001; resubmission received October 24, 2001; revised resubmission received December 3, 2001; accepted December 3, 2001.
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