Polycyclic aromatic hydrocarbon-induced signaling events relevant to inflammation and tumorigenesis in lung cells are dependent on molecular structure.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and occupational toxicants, which are a major human health concern in the U.S. and abroad. Previous research has focused on the genotoxic events caused by high molecular weight PAHs, but not on non-genotoxic events elicited by low molecular weight PAHs. We used an isomeric pair of low molecular weight PAHs, namely 1-Methylanthracene (1-MeA) and 2-Methylanthracene (2-MeA), in which only 1-MeA possessed a bay-like region, and hypothesized that 1-MeA, but not 2-MeA, would affect non-genotoxic endpoints relevant to tumor promotion in murine C10 lung cells, a non-tumorigenic type II alveolar pneumocyte and progenitor cell type of lung adenocarcinoma. The non-genotoxic endpoints assessed were dysregulation of gap junction intercellular communication function and changes in the major pulmonary connexin protein, connexin 43, using fluorescent redistribution and immunoblots, activation of mitogen activated protein kinases (MAPK) using phosphospecific MAPK antibodies for immunoblots, and induction of inflammatory genes using quantitative RT-PCR. 2-MeA had no effect on any of the endpoints, but 1-MeA dysregulated gap junctional communication in a dose and time dependent manner, reduced connexin 43 protein expression, and altered membrane localization. 1-MeA also activated ERK1/2 and p38 MAP kinases. Inflammatory genes, such as cyclooxygenase 2, and chemokine ligand 2 (macrophage chemoattractant 2), were also upregulated in response to 1-MeA only. These results indicate a possible structure-activity relationship of these low molecular weight PAHs relevant to non-genotoxic endpoints of the promoting aspects of cancer. Therefore, our novel findings may improve the ability to predict outcomes for future studies with additional toxicants and mixtures, identify novel targets for biomarkers and chemotherapeutics, and have possible implications for future risk assessment for these PAHs.

Epidermal growth factor regulates hematopoietic regeneration after radiation injury.

The mechanisms that regulate hematopoietic stem cell (HSC) regeneration after myelosuppressive injury are not well understood. We identified epidermal growth factor (EGF) to be highly enriched in the bone marrow serum of mice bearing deletion of Bak and Bax in TIE2-expressing cells in Tie2Cre; Bak1(-/-); Bax(flox/-) mice. These mice showed radioprotection of the HSC pool and 100% survival after a lethal dose of total-body irradiation (TBI). Bone marrow HSCs from wild-type mice expressed functional EGF receptor (EGFR), and systemic administration of EGF promoted the recovery of the HSC pool in vivo and improved the survival of mice after TBI. Conversely, administration of erlotinib, an EGFR antagonist, decreased both HSC regeneration and the survival of mice after TBI. Mice with EGFR deficiency in VAV-expressing hematopoietic cells also had delayed recovery of bone marrow stem and progenitor cells after TBI. Mechanistically, EGF reduced radiation-induced apoptosis of HSCs and mediated this effect through repression of the proapoptotic protein PUMA. Our findings show that EGFR signaling regulates HSC regeneration after myelosuppressive injury.

Tie2(+) bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury.

Hematopoietic stem cells (HSCs) reside in proximity to bone marrow endothelial cells (BM ECs) and maintenance of the HSC pool is dependent upon EC-mediated c-kit signaling. Here, we used genetic models to determine whether radioprotection of BM ECs could facilitate hematopoietic regeneration following radiation-induced myelosuppression. We developed mice bearing deletion of the proapoptotic proteins, BAK and BAX, in Tie2(+) ECs and HSCs (Tie2Bak/Bax(Fl/-) mice) and compared their hematopoietic recovery following total body irradiation (TBI) with mice which retained Bax in Tie2(+) cells. Mice bearing deletion of Bak and Bax in Tie2(+) cells demonstrated protection of BM HSCs, preserved BM vasculature, and 100% survival following lethal dose TBI. In contrast, mice that retained Bax expression in Tie2(+) cells demonstrated depletion of BM HSCs, disrupted BM vasculature, and 10% survival post-TBI. In a complementary study, VEcadherinBak/Bax(Fl/-) mice, which lack Bak and Bax in VEcadherin(+) ECs, also demonstrated increased recovery of BM stem/progenitor cells following TBI compared to mice which retained Bax in VEcadherin(+) ECs. Importantly, chimeric mice that lacked Bak and Bax in HSCs but retained Bak and Bax in BM ECs displayed significantly decreased HSC content and survival following TBI compared to mice lacking Bak and Bax in both HSCs and BM ECs. These data suggest that the hematopoietic response to ionizing radiation is dependent upon HSC-autonomous responses but is regulated by BM EC-mediated mechanisms. Therefore, BM ECs may be therapeutically targeted as a means to augment hematopoietic reconstitution following myelosuppression.

Pleiotrophin regulates the retention and self-renewal of hematopoietic stem cells in the bone marrow vascular niche.

The mechanisms through which the bone marrow (BM) microenvironment regulates hematopoietic stem cell (HSC) fate remain incompletely understood. We examined the role of the heparin-binding growth factor pleiotrophin (PTN) in regulating HSC function in the niche. PTN(-/-) mice displayed significantly decreased BM HSC content and impaired hematopoietic regeneration following myelosuppression. Conversely, mice lacking protein tyrosine phosphatase receptor zeta, which is inactivated by PTN, displayed significantly increased BM HSC content. Transplant studies revealed that PTN action was not HSC autonomous, but rather was mediated by the BM microenvironment. Interestingly, PTN was differentially expressed and secreted by BM sinusoidal endothelial cells within the vascular niche. Furthermore, systemic administration of anti-PTN antibody in mice substantially impaired both the homing of hematopoietic progenitor cells to the niche and the retention of BM HSCs in the niche. PTN is a secreted component of the BM vascular niche that regulates HSC self-renewal and retention in vivo.

Targeted deletion of Nrf2 reduces urethane-induced lung tumor development in mice.

Nrf2 is a key transcription factor that regulates cellular redox and defense responses. However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance. We tested the hypothesis that Nrf2 has cell survival properties and lack of Nrf2 suppresses chemically-induced pulmonary neoplasia by treating Nrf2(+/+) and Nrf2(-/-) mice with urethane. Airway inflammation and injury were assessed by bronchoalveolar lavage analyses and histopathology, and lung tumors were analyzed by gross and histologic analysis. We used transcriptomics to assess Nrf2-dependent changes in pulmonary gene transcripts at multiple stages of neoplasia. Lung hyperpermeability, cell death and apoptosis, and inflammatory cell infiltration were significantly higher in Nrf2(-/-) mice compared to Nrf2(+/+) mice 9 and 11 wk after urethane. Significantly fewer lung adenomas were found in Nrf2(-/-) mice than in Nrf2(+/+) mice at 12 and 22 wk. Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia. In lung tumors, Nrf2-altered genes had roles in transcriptional regulation of cell cycle and proliferation, carcinogenesis, organismal injury and abnormalities, xenobiotic metabolism, and cell-cell signaling genes. Collectively, Nrf2 deficiency decreased susceptibility to urethane-induced lung tumorigenesis in mice. Cell survival properties of Nrf2 were supported, at least in part, by reduced early death of initiated cells and heightened advantage for tumor cell expansion in Nrf2(+/+) mice relative to Nrf2(-/-) mice. Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.