FoxM1 in tumorigenicity of the neuroblastoma cells and renewal of the neural progenitors.

Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma. We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 (Sox2) and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells. By using a conditional deletion system and neurosphere cultures, we showed that FoxM1 is important for expression of Sox2 and Bmi1 in the mouse neural stem/progenitor cells and is critical for its self-renewal. Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state.

Deregulation of FoxM1b leads to tumour metastasis.

The forkhead box M1b (FoxM1b) transcription factor is over-expressed in human cancers, and its expression often correlates with poor prognosis. Previously, using conditional knockout strains, we showed that FoxM1b is essential for hepatocellular carcinoma (HCC) development. However, over-expression of FoxM1b had only marginal effects on HCC progression. Here we investigated the effect of FoxM1b expression in the absence of its inhibitor Arf. We show that transgenic expression of FoxM1b in an Arf-null background drives hepatic fibrosis and metastasis of HCC. We identify novel mechanisms of FoxM1b that are involved in epithelial-mesenchymal transition, cell motility, invasion and a pre-metastatic niche formation. FoxM1b activates the Akt-Snail1 pathway and stimulates expression of Stathmin, lysyl oxidase, lysyl oxidase like-2 and several other genes involved in metastasis. Furthermore, we show that an Arf-derived peptide, which inhibits FoxM1b, impedes metastasis of the FoxM1b-expressing HCC cells. The observations indicate that FoxM1b is a potent activator of tumour metastasis and that the Arf-mediated inhibition of FoxM1b is a critical mechanism for suppression of tumour metastasis.

Negative regulation of the oncogenic transcription factor FoxM1 by thiazolidinediones and mithramycin.

The Forkhead Box transcription factor FoxM1 regulates expression of genes that promote cell cycle progression, and it plays essential roles in the development of liver, lung, prostate and colorectal tumors. Thiazolidinediones (TZDs) activate the peroxisome proliferator-activated receptor gamma (PPARγ), a ligand-activated nuclear receptor transcription factor. We found that treatment of the human hepatoma cell lines HepG2 and PLC/PRF/5 cells with TZDs leads to inhibition of FoxM1 gene expression. No PPARγ/retinoid X receptor (RXR) consensus DNA binding sites were detected in the FoxM1 promoter extending to -10 kb upstream, and knockdown of PPARγ had no impact on TZD mediated downregulation of FoxM1 expression. Previously, others showed that PPARγ agonists inhibit the expression and DNA-binding activity of the Sp1 transcription factor. Here we show that Sp1 binds to the FoxM1 promoter region and positively regulates FoxM1 transcription, while mithramycin, a chemotherapy drug that specifically binds GC rich sequences in the DNA and inhibits activities of Sp1, inhibits expression of FoxM1. Our data suggest that TZD mediated suppression of Sp1 is responsible for downregulation of FoxM1 gene expression. Inhibition of FoxM1 expression by TZDs provides a new mechanism for TZD mediated negative regulation of cancer cell growth. FoxM1 expression and activity in cancer cells can be targeted using PPARγ agonists or the anti-neoplastic antibiotic mithramycin.

A conserved phosphorylation site within the forkhead domain of FoxM1B is required for its activation by cyclin-CDK1.

The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G(1)/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G(2)/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G(2)/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.

Multiple, temporal-specific roles for HNF6 in pancreatic endocrine and ductal differentiation.

Within the developing pancreas Hepatic Nuclear Factor 6 (HNF6) directly activates the pro-endocrine transcription factor, Ngn3. HNF6 and Ngn3 are each essential for endocrine differentiation and HNF6 is also required for embryonic duct development. Most HNF6(-/-) animals die as neonates, making it difficult to study later aspects of HNF6 function. Here, we describe, using conditional gene inactivation, that HNF6 has specific functions at different developmental stages in different pancreatic lineages. Loss of HNF6 from Ngn3-expressing cells (HNF6(Delta endo)) resulted in fewer multipotent progenitor cells entering the endocrine lineage, but had no effect on beta cell terminal differentiation. Early, pancreas-wide HNF6 inactivation (HNF6(Delta panc)) resulted in endocrine and ductal defects similar to those described for HNF6 global inactivation. However, all HNF6(Delta panc) animals survived to adulthood. HNF6(Delta panc) pancreata displayed increased ductal cell proliferation and metaplasia, as well as characteristics of pancreatitis, including up-regulation of CTGF, MMP7, and p8/Nupr1. Pancreatitis was most likely caused by defects in ductal primary cilia. In addition, expression of Prox1, a known regulator of pancreas development, was decreased in HNF6(Delta panc) pancreata. These data confirm that HNF6 has both early and late functions in the developing pancreas and is essential for maintenance of Ngn3 expression and proper pancreatic duct morphology.

FoxM1, a critical regulator of oxidative stress during oncogenesis.

The transcription factor FoxM1 is over-expressed in most human malignancies. Although it is evident that FoxM1 has critical functions in tumour development and progression, the mechanisms by which FoxM1 participates in those processes are not understood. Here, we describe an essential role of FoxM1 in the regulation of oxidative stress that contributes to malignant transformation and tumour cell survival. We identify a negative feedback loop involving FoxM1 that regulates reactive oxygen species (ROS) in proliferating cells. We show that induction of FoxM1 by oncogenic Ras requires ROS. Elevated FoxM1, in turn, downregulates ROS levels by stimulating expression of ROS scavenger genes, such as MnSOD, catalase and PRDX3. FoxM1 depletion sensitizes cells to oxidative stress and increases oncogene-induced premature senescence. Moreover, tumour cells expressing activated AKT1 are 'addicted' to FoxM1, as they require continuous presence of FoxM1 for survival. Together, our results identify FoxM1 as a key regulator of ROS in dividing cells, and provide insights into the mechanism how tumour cells use FoxM1 to control oxidative stress to escape premature senescence and apoptosis.

Beta-cell proliferation, but not neogenesis, following 60% partial pancreatectomy is impaired in the absence of FoxM1.

OBJECTIVE: This study was designed to determine whether the transcription factor FoxM1 was required for regeneration of beta-cell mass via proliferation and/or neogenesis in the adult after 60% partial pancreatectomy (PPx). RESEARCH DESIGN AND METHODS: Adult mice with a pancreas-wide deletion of Foxm1 (Foxm1(flox/flox);Pdx1-Cre [FoxM1(Deltapanc)]) and their control littermates (Foxm1(flox/flox)) were subjected to PPx or a sham operation, after which islet expression of Foxm1 and several target genes, beta-cell mass, proliferation, beta-cell size, islet size, islet density, and neurogenin-3 expression were analyzed. RESULTS: In control mice, PPx stimulated beta-cell proliferation and neogenesis and upregulated Foxm1 and several of its known targets (Plk1, Cenp-a, Birc5/Survivin, and Ccnb1) in islets. Within 1 week post-PPx, control mice underwent significant regeneration of beta-cell mass, and average islet size within the regenerating lobe was similar to that after a sham operation. However, FoxM1(Deltapanc) mice exhibited specific impairments in beta-cell mass regeneration and islet growth after PPx, with reduced proliferation of alpha- and beta-cells but no impairments in acinar or ductal cell proliferation. Interestingly, FoxM1 was not required for proliferation of beta-cells within small endocrine cell clusters located in the regenerating portion of the pancreas but was specifically required for proliferation of beta-cells within larger islets. Additionally, FoxM1 was not required for beta-cell neogenesis following PPx. CONCLUSIONS: Our results indicate that FoxM1 is partially required for increased beta-cell proliferation, but not beta-cell neogenesis, stimulated by PPx. Furthermore, FoxM1 seems to be dispensable for proliferation of beta-cells following neogenesis but is required for proliferation of preexisting beta-cells.

FoxM1 regulates transcription of JNK1 to promote the G1/S transition and tumor cell invasiveness.

The Forkhead box M1 (FoxM1) protein is a proliferation-specific transcription factor that plays a key role in controlling both the G(1)/S and G(2)/M transitions through the cell cycle and is essential for the development of various cancers. We show here that FoxM1 directly activates the transcription of the c-Jun N-terminal kinase (JNK1) gene in U2OS osteosarcoma cells. Expression of JNK1, which regulates the expression of genes important for the G(1)/S transition, rescues the G(1)/S but not the G(2)/M cell cycle block in FoxM1-deficient cells. Knockdown of either FoxM1 or JNK1 inhibits tumor cell migration, invasion, and anchorage-independent growth. However, expression of JNK1 in FoxM1-depleted cells does not rescue these defects, indicating that JNK1 is a necessary but insufficient downstream mediator of FoxM1 in these processes. Consistent with this interpretation, FoxM1 regulates the expression of the matrix metalloproteinases MMP-2 and MMP-9, which play a role in tumor cell invasion, through JNK1-independent and -dependent mechanisms in U2OS cells, respectively. Taken together, these findings identify JNK1 as a critical transcriptional target of FoxM1 that contributes to FoxM1-regulated cell cycle progression, tumor cell migration, invasiveness, and anchorage-independent growth.

Anaphase-promoting complex/cyclosome-CDH1-mediated proteolysis of the forkhead box M1 transcription factor is critical for regulated entry into S phase.

The forkhead box M1 (FoxM1) transcription factor is overexpressed in many cancers, and in mouse models it is required for tumor progression. FoxM1 activates expression of the cell cycle genes required for both S and M phase progression. Here we demonstrate that FoxM1 is degraded in late mitosis and early G(1) phase by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. FoxM1 interacts with the APC/C complex and its adaptor, Cdh1. Expression of Cdh1 stimulated degradation of the FoxM1 protein, and depletion of Cdh1 resulted in stabilization of the FoxM1 protein in late mitosis and in early G(1) phase of the cell cycle. Cdh1 has been implicated in regulating S phase entry. We show that codepletion of FoxM1 inhibits early S phase entry observed in Cdh1-depleted cells. The N-terminal region of FoxM1 contains both destruction box (D box) and KEN box sequences that are required for targeting by Cdh1. Mutation of either the D box sequence or the KEN box sequence stabilized FoxM1 and blocked Cdh1-induced proteolysis. Cells expressing a nondegradable form of FoxM1 entered S phase rapidly following release from M phase arrest. Together, our observations show that FoxM1 is one of the targets of Cdh1 in late M or early G(1) phase and that its proteolysis is important for regulated entry into S phase.

FoxM1 regulates growth factor-induced expression of kinase-interacting stathmin (KIS) to promote cell cycle progression.

The Forkhead box M1 (FoxM1) transcription factor is essential for cell cycle progression and mitosis. FoxM1 regulates expression of Skp2 and Cks1, subunits of the SCF ubiquitin ligase complex, which ubiquitinates p27(Kip1) and targets it for degradation. Kinase-interacting stathmin (KIS) is a growth factor-dependent nuclear kinase that regulates cell cycle progression by phosphorylating p27(Kip1) to promote its nuclear export. Here we present an additional mechanism of FoxM1-mediated regulation of p27(Kip1) and provide evidence that FoxM1 regulates growth factor-induced expression of KIS. In cells harboring FoxM1 deletion or expressing FoxM1-short interfering RNA, the expression of KIS is impaired, leading to an accumulation of p27(Kip1) in the nucleus. Furthermore, we show that KIS is a direct transcriptional target of FoxM1. Thus FoxM1 promotes cell cycle progression by down-regulating p27(Kip1) through multiple mechanisms.

Transplanted hepatocytes over-expressing FoxM1B efficiently repopulate chronically injured mouse liver independent of donor age.

Orthotopic liver transplantation is limited by the shortage of liver donors, leading to elderly patients being enrolled as donors with increasing frequency. Alternative strategies such as cell therapy are therefore needed. Because transplanted hepatocytes do not proliferate into a recipient liver, repopulation strategies have been developed. We have previously published a proof of concept that hepatocytes harboring a survival selective advantage can efficiently repopulate a mouse liver. We develop here an alternative approach by conferring a selective proliferative advantage on transplanted hepatocytes over resident ones. FoxM1B is a transcription factor that, when over-expressed into hepatocytes, accelerates the cell cycle and maintains the hepatocyte in vivo proliferative capacity of aged livers. We now demonstrate that transplanted hepatocytes over-expressing FoxM1B repopulate the liver of mice subjected to continuous injury far more efficiently than control hepatocytes. We show that old hepatocytes that over-express FoxM1B retain their cell division capacity and repopulate liver as well as young ones, in contrast with old non-modified hepatocytes, which lose their proliferative capacity. In conclusion, our results point to the potential use of FoxM1B expression in hepatocyte-based therapy protocols in diseases where host hepatocytes are chronically injured, especially if donor hepatocytes come from old livers.

The forkhead box M1 transcription factor contributes to the development and growth of mouse colorectal cancer.

BACKGROUND & AIMS: In this study, we used Forkhead Box m1b (Foxm1b) transgenic mice and conditional Foxm1 knock-out mice to examine the role of Foxm1 in colon cancer development and proliferation. METHODS: To induce mouse colorectal cancer, we used a single intraperitoneal injection of azoxymethane (AOM) followed by three 1-week cycles of 2.5% dextran sodium sulfate (DSS) water, each cycle separated by 2 weeks. For these colon tumor studies, we used either Rosa26-Foxm1b transgenic mice that ubiquitously expressed the human Foxm1b complementary DNA or mice in which the Foxm1 fl/fl targeted allele was deleted in colonic epithelial cells using the gut-specific Villin-Cre recombinase transgene (Villin-Cre). Colorectal tumor number and bromodeoxyuridine labeling were determined in Rosa26-Foxm1b mice, Villin-Cre Foxm1-/-, mice and wild-type mice after 12 weeks of AOM/DDS exposure. We also used Foxm1 small interfering RNA-depleted human DLD1 and mouse CT26 colon cancer cell lines to examine DNA replication and anchorage-independent growth. RESULTS: After 12 weeks of treatment with AOM/DSS, Rosa26 Foxm1b transgenic mice showed an increase in the number and size of colorectal tumors compared with wild-type mice. Likewise, a significant reduction in the development and growth of colorectal tumors was found in Villin-Cre Foxm1-/- mice compared with Foxm1 fl/fl mice after AOM/DSS treatment, which was associated with decreased expression of cyclin A2, cyclin B1, survivin, and T-cell factor 4 genes. Moreover, Foxm1-depleted colon cancer cell lines showed reduced DNA replication and anchorage-independent growth. CONCLUSIONS: These studies suggest that Foxm1 is critical for the proliferation and growth of colorectal cancer.

Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box M1 transcription factor.

The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 -/- mouse line to demonstrate that Foxm1-deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in Foxm1 -/- hearts were associated with a reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21(Cip1) protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in Tie2-Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the -9,259/-9,288-bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxm1 regulates expression of genes essential for the proliferation of cardiomyocytes during heart development.

Functional polymorphism of the Anpep gene increases promoter activity in the Dahl salt-resistant rat.

We have reported that aminopeptidase N/CD13, which metabolizes angiotensin III to angiotensin IV, exhibits greater renal tubular expression in the Dahl salt-resistant (SR/Jr) rat than its salt-sensitive (SS/Jr) counterpart. In this work, aminopeptidase N (Anpep) genes from SS/Jr and SR/Jr strains were compared. The coding regions contained only silent single nucleotide polymorphisms between strains. The 5' flanking regions also contained multiple single nucleotide polymorphisms, which were analyzed by electrophoretic mobility-shift assay using renal epithelial cell (HK-2) nuclear extracts and oligonucleotides corresponding with single nucleotide polymorphism-containing regions. A unique single nucleotide polymorphism 4 nucleotides upstream of a putative CCAAT/enhancer binding protein motif (nucleotides -2256 to -2267) in the 5' flanking region of the SR/Jr Anpep gene was associated with DNA-protein complex formation, whereas the corresponding sequences in SS rats were not. A chimeric reporter gene containing approximately 4.4 Kb of Anpep 5' flank from the Dahl SR/Jr rat exhibited 2.5- to 3-fold greater expression in HK-2 cells than the corresponding construct derived from the SS strain (P<0.05). Replacing the CCAAT/enhancer binding protein cis-acting element from the SS rat with that from the SR strain increased reporter gene expression by 2.5-fold (P<0.05) and abolished this difference. CCAAT/enhancer binding protein association was confirmed by chromatin immunoprecipitation and correlated with expression, suggesting selection for a functional CCAAT/enhancer binding protein polymorphism in the 5' flank of Anpep in the Dahl SR/Jr rat. These results highlight a possible association of the Anpep gene with hypertension in Dahl rat and raise the prospect that increased Anpep may play a mechanistic role in adaptation to high salt.

Chk2 mediates stabilization of the FoxM1 transcription factor to stimulate expression of DNA repair genes.

The forkhead box M1 (FoxM1) transcription factor regulates expression of cell cycle genes essential for DNA replication and mitosis during organ repair and cancer progression. Here, we demonstrate that FoxM1-deficient (-/-) mouse embryonic fibroblasts and osteosarcoma U2OS cells depleted in FoxM1 levels by small interfering RNA transfection display increased DNA breaks, as evidenced by immunofluorescence focus staining for phosphospecific histone H2AX. FoxM1-deficient cells also exhibit stimulation of p53 transcriptional activity, as evidenced by increased expression of the p21(cip1) gene. FoxM1-deficient cells display reduced expression of the base excision repair factor X-ray cross-complementing group 1 (XRCC1) and breast cancer-associated gene 2 (BRCA2), the latter of which is involved in homologous recombination repair of DNA double-strand breaks. Furthermore, FoxM1 protein is phosphorylated by checkpoint kinase 2 (Chk2) in response to DNA damage. This phosphorylation of FoxM1 on serine residue 361 caused increased stability of the FoxM1 protein with corresponding increased transcription of XRCC1 and BRCA2 genes, both of which are required for repair of DNA damage. These results identify a novel role for FoxM1 in the transcriptional response during DNA damage/checkpoint signaling and show a novel mechanism by which Chk2 protein regulates expression of DNA repair enzymes.

A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment.

The forkhead box m1 (Foxm1) transcription factor is essential for initiation of carcinogen-induced liver tumors; however, whether FoxM1 constitutes a therapeutic target for liver cancer treatment remains unknown. In this study, we used diethylnitrosamine/phenobarbital treatment to induce hepatocellular carcinomas (HCCs) in either WT mice or Arf(-/-)Rosa26-FoxM1b Tg mice, in which forkhead box M1b (FoxM1b) is overexpressed and alternative reading frame (ARF) inhibition of FoxM1 transcriptional activity is eliminated. To pharmacologically reduce FoxM1 activity in HCCs, we subjected these HCC-bearing mice to daily injections of a cell-penetrating ARF(26-44) peptide inhibitor of FoxM1 function. After 4 weeks of this treatment, HCC regions displayed reduced tumor cell proliferation and angiogenesis and a significant increase in apoptosis within the HCC region but not in the adjacent normal liver tissue. ARF peptide treatment also induced apoptosis of several distinct human hepatoma cell lines, which correlated with reduced protein levels of the mitotic regulatory genes encoding polo-like kinase 1, aurora B kinase, and survivin, all of which are transcriptional targets of FoxM1 that are highly expressed in cancer cells and function to prevent apoptosis. These studies indicate that ARF peptide treatment is an effective therapeutic approach to limit proliferation and induce apoptosis of liver cancer cells in vivo.

Identification of a chemical inhibitor of the oncogenic transcription factor forkhead box M1.

The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in a number of different carcinomas, whereas its expression is turned off in terminally differentiated cells. For this reason, FoxM1 is an attractive target for therapeutic intervention in cancer treatment. As a first step toward realizing this goal, in this study, using a high-throughput, cell-based assay system, we screened for and isolated the antibiotic thiazole compound Siomycin A as an inhibitor of FoxM1. Interestingly, we observed that Siomycin A was able to down-regulate the transcriptional activity as well as the protein and mRNA abundance of FoxM1. Consequently, we found that the downstream target genes of FoxM1, such as Cdc25B, Survivin, and CENPB, were repressed. Also, we observed that consistent with earlier reports of FoxM1 inhibition, Siomycin A was able to reduce anchorage-independent growth of cells in soft agar. Furthermore, we found that Siomycin A was able to induce apoptosis selectively in transformed but not normal cells of the same origin. Taken together, our data suggest that FoxM1 inhibitor Siomycin A could represent a useful starting point for the development of anticancer therapeutics.

Endothelial cell-restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury.

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27(Kip1) and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27(Kip1) expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.

Increased expression of hepatocyte nuclear factor 6 stimulates hepatocyte proliferation during mouse liver regeneration.

BACKGROUND & AIMS: The hepatocyte nuclear factor 6 (HNF6 or ONECUT-1) protein is a cell-type specific transcription factor that regulates expression of hepatocyte-specific genes. Using hepatocytes for chromatin immunoprecipitation (ChIP) assays, the HNF6 protein was shown to associate with cell cycle regulatory promoters. Here, we examined whether increased levels of HNF6 stimulate hepatocyte proliferation during mouse liver regeneration. METHODS: Tail vein injection of adenovirus expressing the HNF6 complementary DNA was used to increase hepatic HNF6 levels during mouse liver regeneration induced by partial hepatectomy, and DNA replication was determined by bromodeoxyuridine incorporation. Cotransfection and ChIP assays were used to determine transcriptional target promoters. RESULTS: Elevated expression of HNF6 during mouse liver regeneration causes a significant increase in the number of hepatocytes entering DNA replication (S phase), and mouse hepatoma Hepa1-6 cells diminished for HNF6 levels by small interfering RNA transfection exhibit a 50% reduction in S phase following serum stimulation. This stimulation in hepatocyte S-phase progression was associated with increased expression of the hepatocyte mitogen tumor growth factor alpha and the cell cycle regulators cyclin D1 and Forkhead box m1 (Foxm1) transcription factor. Cotransfection and ChIP assays show that tumor growth factor alpha, cyclin D1, and HNF6 promoter regions are direct transcriptional targets of the HNF6 protein. Coimmunoprecipitation assays with regenerating mouse liver extracts reveal an association between HNF6 and FoxM1 proteins, and cotransfection assays show that HNF6 stimulates Foxm1 transcriptional activity. CONCLUSIONS: These mouse liver regeneration studies show that increased HNF6 levels stimulate hepatocyte proliferation through transcriptional induction of cell cycle regulatory genes.

The FoxM1 transcription factor is required to maintain pancreatic beta-cell mass.

The FoxM1 transcription factor is highly expressed in proliferating cells and activates several cell cycle genes, although its requirement appears to be limited to certain tissue types. Embryonic hepatoblast-specific inactivation of Foxm1 results in a dramatic reduction in liver outgrowth and subsequent late gestation lethality, whereas inactivation of Foxm1 in adult liver impairs regeneration after partial hepatectomy. These results prompted us to examine whether FoxM1 functions similarly in embryonic outgrowth of the pancreas and beta-cell proliferation in the adult. We found that FoxM1 is highly expressed in embryonic and neonatal endocrine cells, when many of these cells are proliferating. Using a Cre-lox strategy, we generated mice in which Foxm1 was inactivated throughout the developing pancreatic endoderm by embryonic d 15.5 (Foxm1(Deltapanc)). Mice lacking Foxm1 in their entire pancreas were born with normal pancreatic and beta-cell mass; however, they displayed a gradual decline in beta-cell mass with age. Failure of postnatal beta-cell mass expansion resulted in impaired islet function by 6 wk of age and overt diabetes by 9 wk. The decline in beta-cell mass in Foxm1(Deltapanc) animals is due to a dramatic decrease in postnatal beta-cell replication and a corresponding increase in nuclear localization of the cyclin-dependent kinase inhibitor, p27(Kip1), a known target of FoxM1 inhibition. We conclude that Foxm1 is essential to maintain normal beta-cell mass and regulate postnatal beta-cell turnover. These results suggest that mechanisms regulating embryonic beta-cell proliferation differ from those used postnatally to maintain the differentiated cell population.