Xiaowei Jia

Benzo(a)pyrene-caused increased G1–S transition requires the activation of c-Jun through p53-dependent PI-3K/Akt/ERK pathway in human embryo lung fibroblasts

Benzo(a)pyrene (B(a)P) is a potent lung carcinogen mainly derived from tobacco smoking and environmental contamination, however, the molecular mechanisms by which it accelerates the cell cycle progression and induces the abnormal cell proliferation are still far away from understood. Our current analysis of human embryo lung fibroblasts (HELF) showed that B(a)P exposure was able to promote cell cycle G(1)-S phase transition. This effect was correlated with c-Jun activation because inhibition of c-Jun by its dominant negative mutant (TAM67) reversed B(a)P action on cell cycle with the down-regulation of expression of cyclin D1, pRb and E2F1. Further study found that overexpression of dominant negative mutants of, PI-3K or Akt, dramatically reduced B(a)P-induced the activation of c-Jun and extracellular signaling regulated kinase (ERK), but not c-Jun NH2 terminal kinase (JNK). Inhibition of p53 by either its inhibitor pifithrin-alpha or p53 siRNA markedly increased B(a)P-induced the activation of c-Jun, Akt and ERK in this context. Take together, our results indicate that c-Jun activation by p53-dependent PI-3K/Akt/ERK pathway is responsible for B(a)P-induced cell cycle alternations in human embryo lung fibroblasts.

Roles of the ERK, JNK/AP-1/cyclin D1-CDK4 pathway in silica-induced cell cycle changes in human embryo lung fibroblast cells.

Silica is a potent occupational fibrogenic agent capable of inducing lung fibrosis and many other lung diseases. Our current study focused on the signalling pathways regulating cell cycle changes in HELF (human embryo lung fibroblast) after silica (α‐quartz) exposure. Our results showed silica exposure could lead to cell cycle changes. The cell cycle alternations were accompanied with overexpression of cyclin D1 and CDK4 (cyclin‐dependent kinase 4) in a time‐dependent manner. Silica exposure also decreased E2F‐4 expression in HELF. These changes were blocked by overexpression of dominant‐negative mutants of ERK (extracellular signal‐regulated protein kinase) or the JNK (stress‐activated c‐Jun NH2‐terminal kinase), respectively. Moreover, pretreatment of cells with curcumin, an activation of AP‐1 (activator protein‐1) inhibitor, inhibited silica‐induced cell cycle alteration, the decreased expression of E2F‐4 and overexpression of cyclin D1 and CDK4. Furthermore, both antisense cyclin D1 and antisense CDK4 can block silica‐induced cell cycle changes. These results suggest that silica exposure can induce cell cycle changes, which may be mediated through ERK, JNK/AP‐1/cyclin D1–CDK4‐dependent pathway.

Inhibition of benzo(a)pyrene-induced cell cycle progression by all-trans retinoic acid partly through cyclin D1/E2F-1 pathway in human embryo lung fibroblasts

Benzo(a)pyrene [B(a)P] is a potent environmental carcinogen, which induces cell cycle changes. All‐trans retinoic acid (ATRA) is a promising agent in prevention and treatment of human cancers. In the present study, we investigated the inhibition of B(a)P‐induced cell cycle progression by ATRA in human embryo lung fibroblast (HELF). Our results showed that after treatment with B(a)P, the expression of cyclin D1 and E2F‐1 were both increased significantly in HELF. There were almost no changes of CDK4 and E2F‐4 expression by treatment with B(a)P. As expected, pretreatment with ATRA could efficiently decrease B(a)P‐induced overexpression of cyclin D1 and E2F‐1. In a further study, we stably transfected antisense cyclin D1 and antisense CDK4 plasmid into HELF. The inhibition of cyclin D1 expression and the inhibition of CDK4 expression significantly impaired the B(a)P‐induced overexpression of E2F‐1 respectively. Pretreatment with ATRA, cells expressing antisense cyclinD1 or antisense CDK4 showed a lesser decrease of B(a)P‐induced overexpression of E2F‐1 compared with similarly treated HELF. Furthermore, flow cytometry analysis showed that B(a)P promoted cell cycle progression from G1 phase to S phase, while pretreatment with ATRA could inhibit B(a)P‐induced cell cycle progression by an accumulation of cells in the G1 phase. It was suggested that ATRA could block B(a)P‐induced cell cycle promotion partly through the cyclin D1/E2F‐1 pathway in HELF.

Downregulation of cyclin D1-CDK4 protein in human embryonic lung fibroblasts (HELF) induced by silica is mediated through the ERK and JNK pathway.

Silica is a factor in the induction of acute injury and chronic pulmonary fibrosis. In 1996, silica was also listed as a human carcinogen by the International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved in its pathologic effects are not well understood. We found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica for 2 h decreased cyclin D1 and cyclin‐dependent kinase 4 (CDK4) expression levels. Extracellular signal‐regulated protein kinase (ERKs), c‐Jun NH2‐terminal amino kinase (JNKs), and p38 kinase, as well as their downstream transcription factor, AP‐1, had different effects on the regulation of expression levels of cyclin D1 and CDK4 alterations induced by silica.

Silica induces cell cycle changes through PI-3K/AP-1 pathway in human embryo lung fibroblast cells.

Exposure to silica is associated with progressive pulmonary inflammation and fibrosis. Our previous study had demonstrated silica exposure could cause cell cycle alternation and activator protein‐1 (AP‐1) activation. This study showed that silica exposure induced phosphorylation of p70S6 kinase (p70S6K) and Akt in human embryo lung fibroblasts (HELFs). These changes were blocked by overexpression of dominant‐negative mutants of phosphatidylinositol‐3 kinase (Δp85) or Akt (DN‐Akt), respectively. Moreover, pretreatment of cells with rapamycin, a specific p70S6K inhibitor, could inhibit silica‐induced cell cycle alteration, AP‐1 activation, and phosphorylation of p70S6K, but had no effect on Akt phosphorylation. This suggested that phosphatidylinositol‐3 kinase (PI‐3K)/AP‐1 pathway was likely responsible for cell cycle changes. Furthermore, we observed the effect of the pathway on cell cycle regulatory proteins. Our results indicated that inactivation of PI‐3K, Akt, or p70S6K could inhibit silica‐induced overexpression of cyclin D1 and cyclin‐dependent kinase 4 (CDK4) and decreased expression of E2F‐4. Taken together, silica could induce cell cycle changes through PI‐3K/ AP‐1 pathway in HELFs. Copyright

Different Patterns of Cyclin D1/CDK4-E2F-1/4 Pathways in Human Embryo Lung Fibroblasts Treated by Benzo[a]pyrene at Different Doses

OBJECTIVE To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]P. METHODS Human embryo lung fibroblasts (HELFs) were treated with 2 micromol/L or 100 micromol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs). Cyclin D1, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle. RESULTS After B[a]P treatment, the proportion of the first gap (G1) phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 micromol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs (A-D1 and A-K4) and T-HELFs (T-A-D1 and T-A-K4). After 2 micromol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4. CONCLUSIONS Cyclin D1/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 micromol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 micromol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.