Research Article: Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability

Date Published: July 27, 2017

Publisher: Public Library of Science

Author(s): Michela Zago, Jared A. Sheridan, Hussein Traboulsi, Emelia Hecht, Yelu Zhang, Necola Guerrina, Jason Matthews, Parameswaran Nair, David H. Eidelman, Qutayba Hamid, Carolyn J. Baglole, Sanjay B. Maggirwar.


Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

Partial Text

Cigarette smoke is the leading risk factor for chronic obstructive pulmonary disease (COPD), an obstructive lung disease typified by the increased expression of inflammatory mediators such as interleukin-1 (IL-1) and cyclooxygenase-2 (COX-2) [1, 2]. COX-2 is an immediate/early gene that catalyzes the transformation of arachidonic acid (AA) into thromboxanes and prostaglandins (PG) such as PGE2. Inhibition of COX-2-derived PGE2 protects against the development of emphysema [3] which supports a role for chronic COX-2/PGs in the pathobiology of COPD.

COPD is an obstructive lung disease that is increasing in prevalence worldwide, affecting an estimated 200 million people [29]. While the etiology of COPD is strongly linked to smoke exposure, the underlying pathogenic mechanisms by which smoke causes chronic, aberrant pulmonary inflammation remains poorly defined. The purpose of this study was to further understand how the AhR suppresses COX-2 expression in association with COPD. We have published that two signalling pathways involving the NF-κB protein RelB and the AhR diminish the expression of inflammatory mediators, including COX-2, caused by cigarette smoke exposure [8, 30]. We had postulated that AhR-dependent induction of miR-146a serves as a post-transcriptional regulatory mechanism for the attenuation of COX-2 protein expression. A significant component of this was based on our intriguing observations that lung fibroblasts from COPD subjects expressed significantly more basal COX-2 protein compared to fibroblasts derived from either At Risk or Normal subjects (Fig 1). This result is consistent with a report by Togo and colleagues who demonstrated heightened COX-2 protein expression in COPD lung fibroblasts compared to fibroblasts derived from smokers [31]. We extended this finding by providing further evidence that this increase in COX-2 is an inherent feature not due to smoke exposure alone, as there was no difference in COX-2 between cells from Normal subjects compared to At Risk subjects. These data also highlight that the heightened COX-2 protein in COPD-derived lung fibroblasts was not the result of heightened Cox-2 mRNA expression (Fig 1), implying that basal COX-2 protein levels in COPD lung cells are controlled by mechanisms independent from direct transcriptional regulation. To understand the basis of this, we turned our attention to the AhR, as we have recently shown there is less AhR protein in COPD lung fibroblasts [9]. Using complementary techniques in lung structural cells, we confirmed that loss of AhR expression contributes to increased COX-2 protein without a concomitant increase in Cox-2 mRNA levels, supporting the notion that homeostatic control over COX-2 protein- in the absence of exogenous inflammatory stimuli- is dependent on AhR expression.




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