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. 2016 Dec;55(6):889-898.
doi: 10.1165/rcmb.2015-0237OC.

Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium

Pankita H Pandya  1   2 , Amanda J Fisher  2 , Elizabeth A Mickler  2 , Constance J Temm  3 , Kelsey P Lipking  3 , Adam Gracon  2 , Katia Rothhaar  1   4 , George E Sandusky  3 , Mary Murray  5 , Karen Pollok  5 , Jamie Renbarger  5 , Janice S Blum  1   2 , Tim Lahm  2   4 , David S Wilkes  1   2   4
Affiliations

Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium

Pankita H Pandya et al. Am J Respir Cell Mol Biol. 2016 Dec.

Abstract

Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium.

Keywords: complement regulatory proteins; hypoxia; lung.

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Figures

Figure 1.
Figure 1.
Hypoxia induces hypoxia-inducible factor (HIF)-1α in small-airway epithelial cells (SAECs) and down-regulates CD55. SAECs were exposed to hypoxia (1% O2) at times shown. Normoxic (21% O2) cells were controls. (A) Hypoxic SAECs were probed for HIF-1α. Lamin β1 served as an internal loading control. (B) Densitometry analysis indicated significant up-regulation of HIF-1α by 6 hours of hypoxia versus normoxic untreated (0 hour controls) conditions. Data are representative of means (±SEM); n = 5; *P < 0.05 versus normoxia; one-way ANOVA with Bonferroni posttest. (C) CA9 transcripts increased with hypoxia exposure compared with normoxia. Data represent means (±SEM); n = 3; *P < 0.05 or **P < 0.01 versus normoxia; one-way ANOVA with Bonferroni posttest. (D) CD55 transcripts were significantly down-regulated by 6 hours of hypoxia compared with the controlled normoxic conditions. Values represent means (±SEM); n = 3; *P < 0.05 versus normoxia; one-way ANOVA with Bonferroni posttest. (E) Hypoxic SAECs were probed for CD55. Vinculin was the internal loading control. (F) Densitometry analysis shows significant down-regulation of CD55 by 72 hours of hypoxic versus normoxic conditions. Data represent means (±SEM); n = 3; *P < 0.05 versus normoxia; one-way ANOVA with Bonferroni posttest. A.U., absorbance units.
Figure 2.
Figure 2.
Silencing HIF-1α in hypoxic SAECs recovers CD55. (A) HIF-1α is silenced in 6-hour hypoxic SAECs transfected with 50 nM HIF-1α small interfering RNA (siRNA) versus 6-hour hypoxic SAECs with control siRNA (50 nM) or in normoxia. Lamin β1 was the loading control. (B) HIF-1α was repressed in 24-hour hypoxic SAECs transfected with HIF-1α siRNA versus control siRNA or normoxic levels. Data represent means (±SEM); n = 3; *P < 0.05 versus normoxia or control siRNA in 24 hours of hypoxia; one-way ANOVA with Bonferroni posttest. (C) CA9 transcripts were repressed in HIF-1α siRNA–transfected 6-hour hypoxic SAECs versus 6-hour hypoxic SAECs with or without control siRNA. Values represent means (±SEM); n = 3; **P < 0.01 versus nomoxia, 6 hours of hypoxia, or control siRNA; one-way ANOVA with Bonferroni posttest. (D) CD55 transcripts were recovered in HIF-1α siRNA–transfected 6-hour hypoxic SAECs versus 6-hour hypoxic SAECs in the presence or absence of control siRNA. Values represent means (±SEM); n = 3; *P < 0.05 versus nomoxia, 6 hours of hypoxia, or control siRNA; one-way ANOVA with Bonferroni posttest. (E) CD55 was assessed in normoxic, 24-hour hypoxic HIF-1α siRNA, or control siRNA–transfected SAECs. Vinculin was the loading control. (F) CD55 was recovered in 24-hour HIF-1α siRNA SAECs versus control siRNA or normoxic levels. Values represent means (±SEM); n = 3; *P < 0.05 versus normoxia, 6 hours of hypoxia, or control siRNA; one-way ANOVA with Bonferroni posttest.
Figure 3.
Figure 3.
Stabilization of HIF-1α in normoxia by dimethyloxaloylglycine (DMOG) results in CD55 down-regulation in SAECs. (A) DMOG-treated (1 μM) SAECs were probed for HIF-1α protein expression. Lamin β1 was used as internal loading control. (B) Densitometry analysis shows HIF-1α induction within 24-hour DMOG (1 μM)-treated SAECs compared with cells with no DMOG. Values represent means (±SEM); n = 3; *P < 0.05 versus nontreated cells (0 hour controls); one-way ANOVA with Bonferroni posttest. (C) CD55 transcripts were down-regulated in 6-hour DMOG (1 μM)-treated SAECs compared with the nontreated cells (0 hour controls). Data represent means (±SEM); n > 3; **P < 0.01 versus 0 hour (nontreated cells); one-way ANOVA with Bonferroni posttest. (D) CD55 was probed for in DMOG (1 μM)-treated SAECs. Lamin β1 was used as internal loading control. (E) Densitometry analysis shows CD55 down-regulation by 24 hours of DMOG (1 μM)-treated SAECs compared with the nontreated cells (0 hour controls). Values represent means (±SEM); n = 3; **P < 0.01 versus nontreated cells (0 hour controls); one-way ANOVA with Bonferroni posttest. (F) CA9 transcripts were up-regulated in DMOG (1 μM)-treated SAECs compared with nontreated cells. Data represent means (±SEM); n = 3; *P < 0.05, or **P < 0.01 versus nontreated cells (0 hour controls); one-way ANOVA with Bonferroni posttest.
Figure 4.
Figure 4.
In vivo, hypoxia results in CD55 down-regulation in mouse lungs. (A) CD55 transcript levels were significantly down-regulated in 24-hour hypoxic mouse lungs compared with lungs of normal mice. Values represent means (±SEM); n > 6; **P < 0.01 versus normal mice; two-tailed unpaired t test. (B) CD55 expression was probed for CD55 in lung homogenates of 24-hour normal and hypoxic mice; β-actin was used as the loading control. (C) Densitometry analysis depicts statistically significant down-regulation of CD55 in 24-hour hypoxic mouse lungs compared with normal mice. Data represent means (±SEM); n = 4; **P < 0.01 versus normal mice; two-tailed unpaired t test. (D) Immunohistochemistry (IHC) analysis shows CD55 down-regulation in 24-hour hypoxic mouse airways compared with control normal mice. Original magnification: ×20. (E) HIF-1α–dependent gene, CA9, was transcriptionally induced in lungs of 24-hour hypoxic mice compared with normal mice. Values represent means (±SEM); n > 4; *P < 0.05 versus normal mice; two-tailed unpaired t test.
Figure 5.
Figure 5.
CD55 expression recovered in hypoxic mouse lungs in which HIF-1α was silenced. (A) CD55 expression was probed for in lung homogenates of 24-hour hypoxic mice intratracheally instilled with HIF-1α siRNA (50 μg) or control siRNA (50 μg); β-actin was used as internal loading control. (B) Densitometry analysis indicated that CD55 was restored significantly in lung homogenates of 24-hour hypoxic mice intratracheally instilled with HIF-1α siRNA (50 μg) compared with control siRNA (50 μg). Data represent means (±SEM); n = 4; *P < 0.05 versus control siRNA; two-tailed unpaired t test. (C) IHC analysis show CD55 down-regulation in hypoxic mouse lungs in which HIF-1α is silenced compared with hypoxic mouse lungs with control siRNA. Original magnification: ×20.
Figure 6.
Figure 6.
Induction of HIF-1α results in CD55 down-regulation. (A) CD55 transcript levels were significantly down-regulated in 6-hour lung homogenates of mice intratracheally instilled with DMOG (1 mg/ml) compared with vehicle controls. Data represent means (±SEM); n = 3; **P < 0.01 versus vehicle controls; two-tailed unpaired t test. (B) CD55 expression was probed in mouse lung homogenates 6 hours after intratracheal instillation with DMOG (1 mg/ml) or vehicle controls. Vinculin was used as internal loading control. (C) Densitometry analysis indicated significant CD55 down-regulation in lung homogenates intratracheally instilled with DMOG (1 mg/ml) or vehicle controls. Values represent means (±SEM); n = 4; **P < 0.01 versus vehicle controls; two-tailed unpaired t test. (D) IHC shows CD55 down-regulation within 6 hours after DMOG instillation versus vehicle control airways. Original magnification: ×20.
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