Neutrophil elastase inhibition of cell cycle progression in airway epithelial cells in vitro is mediated by p27^kip1

Abstract

Neutrophil elastase (NE), a serine protease present in high concentrations in the airways of cystic fibrosis patients, injures the airway epithelium. We examined the epithelial response to NE-mediated proteolytic injury. We have previously reported that NE treatment of airway epithelial cells causes a marked decrease in epithelial DNA synthesis and proliferation. We hypothesized that NE inhibits DNA synthesis by arresting cell cycle progression. Progression through the cell cycle is positively regulated by cyclin complexes and negatively regulated by cyclin-dependent kinase inhibitors (CKI). To test whether NE arrests cell cycle progression, we treated normal human bronchial epithelial (NHBE) cells with NE (50 nM) or control vehicle for 24 h and assessed the effect of treatment on the cell cycle by flow cytometry. NE treatment resulted in G1 arrest. Arrest in G1 phase may be the result of CKI inhibition of the cyclin E complex; therefore, we evaluated whether NE upregulated CKI expression and/or affected the interaction of CKIs with the cyclin E complex. Following NE or control vehicle treatment, expression of p27Kip1, a member of the Cip/Kip family, was evaluated. NE increased p27Kip1 gene and protein expression. NE increased the coimmunoprecipitation of p27Kip1 with cyclin E complex, suggesting that p27Kip1 inhibited cyclin E complex activity. Our results demonstrate that p27 is regulated by NE and is critical for NE-induced cell cycle arrest.

Increased expression of senescence markers in cystic fibrosis airways

“…Thus it is likely that NE exposure contributes to ROS production, oxidative stress, and development of senescence in CF airways. Therefore, NE activates several mechanisms by which it interrupts normal epithelial homeostasis and/or cell fate, namely basal cell hyperplasia and airway epithelial hypertrophy (43), increased epithelial permeability (28), cell cycle arrest (15), apoptosis (16,37), and senescence (Fig. 6).…”

“…NHBE cells were treated with NE (0, 200, or 500 nM) or control vehicle, apically and basolaterally, for 2-5 h until visible injury occurred, and then lysates were collected as previously described (15 (15).…”

“…NE, in concert with increased levels of chemokines present in the bronchoalveolar lavage fluid of patients with CF (4), can lead to airway epithelial injury. We have previously reported that NE induces airway epithelial cell cycle arrest or quiescence (15). Quiescence is a reversible process and cells may re-enter the cell cycle at a later time.…”

Application of mRNA regulatory regions to improve tumor specificity of transgene expression

“…Expression of Rluc linked to 3SV40 was not altered in primary human bronchial epithelial cells after growth supplement deprivation (which leads to partial depletion of cells in S and G 2 /M phases). 8 However, the expression tends to drop in growth supplement-deprived cells when the transgene was linked to DNMT1 3 0 -UTR (Figure 1c). This confirms DNMT1 3 0 -UTR-dependent suppression of transgene expression in G 0 /G 1 (i.e., non-dividing) cells, even the quiescence in the experimental settings used is known not to be complete.…”

“…This confirms DNMT1 3 0 -UTR-dependent suppression of transgene expression in G 0 /G 1 (i.e., non-dividing) cells, even the quiescence in the experimental settings used is known not to be complete. 8 Thus, along with the capacity to specifically stabilize exogenous transcripts only in dividing cells, DNMT1 3 0 -UTR and to the lesser extent TOP2A 3 0 -UTR are capable to support efficient transgene expression at the level comparable to that achieved with traditionally used polyadenylation signals.…”