Cigarette Smoke Induces Human Airway Epithelial Senescence via Growth Differentiation Factor 15 Production.

Cigarette smoke (CS)-induced airway epithelial senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), although the underlying mechanisms remain largely unknown. Growth differentiation factor (GDF) 15 is increased in airway epithelium of smokers with COPD and CS-exposed human airway epithelial cells, but its role in CS-induced airway epithelial senescence is unclear. In this study, we first analyzed expression of GDF15 and cellular senescence markers in airway epithelial cells of current smokers and nonsmokers. Second, we determined the role of GDF15 in CS-induced airway epithelial senescence by using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 genome editing approach. Finally, we examined whether exogenous GDF15 protein promoted airway epithelial senescence through the activin receptor-like kinase 1/Smad1 pathway. GDF15 up-regulation was found in parallel with increased cellular senescence markers, p21, p16, and high-mobility group box 1 in airway epithelial cells of current smokers compared with nonsmokers. Moreover, CS extract induced cellular senescence in cultured human airway epithelial cells, represented by induced senescence-associated ß-galactosidase activity, inhibited cell proliferation, increased p21 expression, and increased release of high-mobility group box 1 and IL-6. Disruption of GDF15 significantly inhibited CS extract-induced airway epithelial senescence. Lastly, GDF15 protein bound to the activin receptor-like kinase 1 receptor and promoted airway epithelial senescence via activation of the Smad1 pathway. Our findings highlight an important contribution of GDF15 in promoting airway epithelial senescence upon CS exposure. Senescent airway epithelial cells that chronically accumulate in CS-exposed lungs could contribute substantially to chronic airway inflammation in COPD development and progression.

Compound haploinsufficiencies of Ebf1 and Runx1 genes impede B cell lineage progression.

Early B cell factor (EBF)1 is essential for B lineage specification. Previously, we demonstrated the synergistic activation of Cd79a (mb-1) genes by EBF1 and its functional partner, RUNX1. Here, we identified consequences of Ebf1 haploinsufficiency together with haploinsufficiency of Runx1 genes in mice. Although numbers of "committed" pro-B cells were maintained in Ebf1(+/-)Runx1(+/-) (ER(het)) mice, activation of B cell-specific gene transcription was depressed in these cells. Expression of genes encoding Aiolos, kappa0 sterile transcripts, CD2 and CD25 were reduced and delayed in ER(het) pro-B cells, whereas surface expression of BP-1 was increased on late pro-B cells in ER(het) mice. Late pre-B and immature and mature B cells were decreased in the bone marrow of Ebf1(+/-) (E(het)) mice and were nearly absent in ER(het) mice. Although we did not observe significant effects of haploinsuficiencies on IgH or Igkappa rearrangements, a relative lack of Iglambda rearrangements was detected in E(het) and ER(het) pre-B cells. Together, these observations suggest that B cell lineage progression is impaired at multiple stages in the bone marrow of E(het) and ER(het) mice. Furthermore, enforced expression of EBF1 and RUNX1 in terminally differentiated plasmacytoma cells activated multiple early B cell-specific genes synergistically. Collectively, these studies illuminate the effects of reduced Ebf1 dosage and the compounding effects of reduced Runx1 dosage. Our data confirm and extend the importance of EBF1 in regulating target genes and Ig gene rearrangements necessary for B cell lineage specification, developmental progression, and homeostasis.

Iloprost requires the Frizzled-9 receptor to prevent lung cancer.

Prevention of premalignant lesion progression is a promising approach to reducing lung cancer burden in high-risk populations. Substantial preclinical and clinical evidence has demonstrated efficacy of the prostacyclin analogue iloprost for lung cancer chemoprevention. Iloprost activates peroxisome proliferator-activated receptor gamma (PPARG) to initiate chemopreventive signaling and in vitro, which requires the transmembrane receptor Frizzled9 (FZD9). We hypothesized a Fzd 9 -/- mouse would not be protected by iloprost in a lung cancer model. Fzd 9 -/- mice were treated with inhaled iloprost in a urethane model of lung adenoma. We found that Fzd 9 -/- mice treated with iloprost were not protected from adenoma development compared to wild-type mice nor did they demonstrate increased activation of iloprost signaling pathways. Our results established that iloprost requires FZD9 in vivo for lung cancer chemoprevention. This work represents a critical advancement in defining iloprost's chemopreventive mechanisms and identifies a potential response marker for future clinical trials.

The 'zinc knuckle' motif of Early B cell Factor is required for transcriptional activation of B cell-specific genes.

Early B cell factor (EBF) is a critical regulator of B lymphocyte-specific gene transcription. EBF functions, in part, by binding to regulatory sites of genes required for the pre-B- and mature B cell receptors. These DNA targets include the promoters of the mb-1 and Vpreb1 genes that encode Ig-alpha and one of the components of surrogate light chain, respectively. The biochemical basis of DNA binding and gene activation by EBF is poorly understood. The DNA-binding domain (DBD) of EBF includes a putative zinc-binding motif (HX(3)CX(2)CX(5)C), which we have designated the 'Zn-knuckle'. The Zn-knuckle is required for binding of the mb-1 promoter site in EMSA, but it has not been demonstrated to be important for functional activities of EBF in B cells. Therefore, we expressed EBF with mutations in the Zn-knuckle motif or flanking sequences in plasmacytoma cells in which activation of endogenous mb-1 and Vpreb1 genes is dependent on EBF. EBF with mutations that prevent zinc coordination by the Zn-knuckle did not activate transcription of either target gene. Other mutations affected the sequence preference of DNA binding and differentially inhibited activation of these genes. Our results demonstrate the importance of the Zn-knuckle motif in EBF. These experiments also confirm that EBF can re-activate multiple genes of the early B cell program in plasmacytoma cells, which provide a useful cell-based assay for dissecting mechanisms involving EBF.