Xinhe Wang

The Forkhead Box m1b transcription factor is essential for hepatocyte DNA replication and mitosis during mouse liver regeneration

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and whose members play essential roles in cellular proliferation, differentiation, transformation, longevity, and metabolic homeostasis. Liver regeneration studies with transgenic mice demonstrated that FoxM1B regulates the onset of hepatocyte DNA replication and mitosis by stimulating expression of cell cycle genes. Here, we demonstrate that albumin-promoter-driven Cre recombinase-mediated hepatocyte-specific deletion of the Foxm1b Floxed (fl) targeted allele resulted in significant reduction in hepatocyte DNA replication and inhibition of mitosis after partial hepatectomy. Reduced DNA replication in regenerating Foxm1b−/− hepatocytes was associated with sustained increase in nuclear staining of the cyclin-dependent kinase (Cdk) inhibitor p21Cip1 (p21) protein between 24 and 40 h after partial hepatectomy. Furthermore, increased nuclear p21 levels and reduced expression of Cdc25A phosphatase coincided with decreases in Cdk2 activation and hepatocyte progression into S-phase. Moreover, the significant reduction in hepatocyte mitosis was associated with diminished mRNA levels and nuclear expression of Cdc25B phosphatase and delayed accumulation of cyclin B1 protein, which is required for Cdk1 activation and entry into mitosis. Cotransfection studies demonstrate that FoxM1B protein directly activated transcription of the Cdc25B promoter region. Our present study shows that the mammalian Foxm1b transcription factor regulates expression of cell cycle proteins essential for hepatocyte entry into DNA replication and mitosis.

Increased levels of forkhead box M1B transcription factor in transgenic mouse hepatocytes prevent age-related proliferation defects in regenerating liver

The forkhead box (Fox) family of transcription factors share homology in the winged helix/forkhead DNA-binding domain and play important roles in regulating cellular proliferation, differentiation, longevity, and cellular transformation. Forkhead box M1B (FoxM1B) is a ubiquitously expressed member of the Fox transcription factor family whose expression is restricted to proliferating cells and that mediates hepatocyte entry into DNA synthesis and mitosis during liver regeneration. Recent cDNA microarray studies indicated that age-related defects in cellular proliferation are associated with diminished expression of the FoxM1B transcription factor. Here, we show that increased levels of FoxM1B in regenerating liver of old transgenic mice restore the sharp peaks in hepatocyte DNA replication and mitosis that are the hallmarks of young regenerating mouse liver. Restoration of the young regenerating liver phenotype is associated with increased expression of numerous cell cycle regulatory genes that include cyclin D1, cyclin A2, cyclin F, cyclin B1, cyclin B2, Cdc25B, and p55cdc. Cotransfection assays in the human hepatoma HepG2 cell line demonstrated that FoxM1B protein stimulated expression of both the cyclin B1 and cyclin D1 promoters, suggesting that these cyclin genes are a direct FoxM1B transcriptional target. These results suggest that FoxM1B controls the transcriptional network of genes that are essential for cell division and exit from mitosis. Our results indicate that reduced expression of the FoxM1B transcription factor contributes to the decline in cellular proliferation observed in the aging process.

Ubiquitous Expression of the Forkhead Box M1B Transgene Accelerates Proliferation of Distinct Pulmonary Cell Types following Lung Injury

The delayed early transcription factor Forkhead Box M1B (FoxM1B) is expressed in proliferating cells, but its expression is extinguished in cells undergoing terminal differentiation. Liver regeneration studies with genetically altered mice that either prematurely expressed FoxM1B in hepatocytes or contained a hepatocyte-specific deletion of the Foxm1b allele demonstrated that FoxM1B is critical for regulating the expression of cell cycle genes required for hepatocyte proliferation. Furthermore, preventing the decline in hepatocyte FoxM1B levels during aging was sufficient to increase regenerating hepatocyte proliferation and expression of cell cycle genes to levels found in young regenerating mouse liver. Although these liver regeneration studies demonstrated that FoxM1B is required for hepatocyte proliferation, whether FoxM1B regulates proliferation of cell types other than hepatocytes remains to be determined. Here, we developed a new TG mouse line in which the –800-base pair Rosa26 promoter was used to drive expression of the FoxM1B transgene in all mouse tissues and found that Rosa26-FoxM1B TG mice were healthy, displaying no developmental defects. We used butylated hydroxytoluene (BHT) lung injury to demonstrate that premature expression of the FoxM1B transgene protein accelerated proliferation of different lung cell types, including alveolar type II epithelial cells, bronchial epithelial and smooth muscle cells, and endothelial cells of pulmonary capillaries and arteries. This was associated with the earlier expression of the cell cycle promoting cyclin A2, cyclin E, cyclin B1, cyclin F, and cyclin dependent kinase-1 (Cdk1) genes and diminished protein levels of Cdk inhibitor p21Cip1. Taken together, these results suggest that increasing FoxM1B levels is an effective means to stimulate cellular proliferation during aging and in lung diseases such as emphysema.

Rapid hepatocyte nuclear translocation of the Forkhead Box M1B (FoxM1B) transcription factor caused a transient increase in size of regenerating transgenic hepatocytes.

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA binding domain. Liver regeneration studies with the -3 kb transthyretin (TTR) promoter-driven FoxM1B transgenic (TG) mice demonstrated that premature hepatocyte nuclear localization of the FoxM1B transgene protein at 16 h following partial hepatectomy (PHx) caused an 8-h acceleration in the onset of hepatocyte DNA replication (S-phase) and mitosis by stimulating earlier expression of cell cycle genes. Whether the FoxM1B transgene protein participates in immediate early events during liver regeneration remains to be determined. Here, we found that the FoxM1B transgene protein translocated to hepatocyte nuclei immediately following PHx, that its nuclear staining persisted for the first 6 h after surgery, and that this translocation was associated with an increase in size of regenerating TG hepatocytes. However, regenerating TTR-FoxM1B liver did not exhibit altered expression of proteins that have been implicated in mediating increased cell size, including serum-and-gucocorticoid-inducible protein kinase (SGK), protein kinase-B/Akt, the tumor suppresser gene PTEN (negative regulator of the PI3K/Akt pathway), c-Myc, or peroxisome proliferation. Moreover, we demonstrated that hepatocyte nuclear translocation of the FoxM1B transgene protein was rapidly induced during the hepatic acute phase response, which occurs during the immediate early stages of liver regeneration. Analysis of cDNA expression arrays identified a number of genes such as immediate early transcription factors (ID-3, Stat3, Nur77), matrix metalloproteinase-9 (MMP-9), and several glutathione S-transferase (GST) isoforms and stress response genes, whose expression is elevated in regenerating TTR-FoxM1B TG livers compared with regenerating wild-type (WT) liver. These liver regeneration studies demonstrate that hepatocyte nuclear translocation of the FoxM1B transgene protein was sustained for the first 6 h after PHx, and was associated with transient hypertrophy of regenerating TG hepatocytes and increased expression of genes that may enhance hepatocyte proliferation.