Therapeutic Effects of Perilla Phenols in Oral Squamous Cell Carcinoma.

The herbal medicine perilla leaf extract (PLE) exhibits various pharmacological properties. We showed that PLE inhibits the viability of oral squamous cell carcinoma (OSCC) cells. HPLC analysis revealed that caffeic acid (CA) and rosmarinic acid (RA) are the two main phenols in PLE, and reduced OSCC cell viability in a dose-dependent manner. The optimal CA/RA combination ratio was 1:2 at concentrations of 300-500 µM but had no synergistic inhibitory effect on the viability of OSCC cells. CA, RA, or their combination effectively suppressed interleukin (IL)-1ß secretion by OSCC OC3 cells. Long-term treatment with CA and CA/RA mixtures, respectively, induced EGFR activation, which might cause OC3 cells to become EGFR-dependent and consequently increased the sensitivity of OC3 cells to a low dose (5 µM) of the EGFR tyrosine kinase inhibitor gefitinib. Chronic treatment with CA, RA, or their combination exhibited an inhibitory effect more potent than that of low-dose (1 µM) cisplatin on the colony formation ability of OSCC cells; this may be attributed to the induction of apoptosis by these treatments. These findings suggest that perilla phenols, particularly CA and RA, can be used as adjuvant therapies to improve the efficacy of chemotherapy and EGFR-targeted therapy in OSCC.

The Buccally Displaced Palatal (BDP) Flap to Increase Quantity and Thickness of Peri-implant Soft Tissue.

Common challenges encountered for atrophic maxilla rehabilitation are the inadequate width and height of attached keratinized mucosa (AKM) and shallow vestibular depth. This study presents a buccally displaced palatal (BDP) flap technique to increase the tissue thickness and AKM width at the second-stage surgery and reestablish the correct fornix depth. The peri-implant pocket depths, modified Plaque Index score, modified sulcus Bleeding Index score, and soft tissue recession were evaluated 6 and 12 months after prostheses loading. A total of 52 implants were placed and analyzed, and no implant failures were found. No significant changes in peri-implant parameters were observed between 6 and 12 months, and mean recession was less than 0.2 mm after 12 months. Though this change was statistically significant, it was clinically irrelevant. The results demonstrate that adequately healthy peri-implant soft tissues and substantial dimensional stability of vestibular soft tissues at the 1-year follow-up were achieved with the BDP flap technique. The BDP flap could represent a viable option for increasing the width and the height of AKM and establishing the correct maxillary fornix depth.

Association between peri-implantitis and cardiovascular diseases: A case-control study.

BACKGROUND: This study assesses the association between peri-implantitis and cardiovascular diseases (CVD). METHODS: One hundred and twenty-eight patients with dental implants were recruited to evaluate the prevalence of peri-implantitis in patients with or without CVD (CVD group, n = 82, control group, n = 46, respectively). Diagnosis of peri-implantitis followed the 2017 World Workshop guidelines and the severity was defined as mild, moderate, and severe form when the radiographic bone loss (RBL) was <2, 2 to 4, and >4 mm. Multivariable logistic regression was performed to test the association between two diseases. RESULTS: A trend of higher prevalence of peri-implantitis defined by detectable RBL beyond the physiologic bone remodeling was found in the CVD group (64.6%) when compared with the controls (56.5%). A significant higher prevalence (48.8%) of moderate to severe peri-implantitis was identified in CVD compared with controls(30.4%) with a significant crude association between moderate to severe peri-implantitis and CVD (odds ratio = 2.18, 95% CI, 1.02 to 4.67; P = 0.04). The CVD group had a trend of higher prevalence of deep pockets (≥7 mm) and higher numbers of sites with bleeding on probing  (>66%) when compared with controls (P > 0.05). However, after controlling for multiple confounders including age, hypertension, smoking, family history of heart attack, and periodontitis, the significant association was not found. CONCLUSIONS: CVD group had significantly higher prevalence of moderate to severe peri-implantitis (RBL ≥2 mm). The association between the two diseases did not exist after controlling multiple confounders for CVD. Future studies with a larger sample size controlling for the patient- and implant-related confounders are needed to better understand the link between peri-implantitis and CVD.

Pro-inflammatory profiles in cardiovascular disease patients with peri-implantitis.

BACKGROUND: To investigate the pro-inflammatory cytokine profiles in patients with or without cardiovascular disease (CVD) and with or without peri-implantitis. METHODS: Serum, peri-implant crevicular fluid (PICF), and gingival crevicular fluid (GCF) were collected from patients with (n = 82) or without CVD (n = 46) at the most severe peri-implantitis site including sites with periodontitis. A panel of proinflammatory molecules including high-sensitivity C-reactive protein (hsCRP), fibrinogen, interleukin-1 beta (IL-1ß), IL-6, plasma tumor necrosis factor-alpha (TNF-α), matrix metallo-proteinase-8 (MMP-8), osteoprotegerin (OPG), vascular endothelial growth factor (VEGF), IL-17, IL-8, tissue inhibitor of metalloproteinase-2 (TIMP-2), myeloperoxidase (MPO), and prostaglandin E2 (PGE2 ) were analyzed using human custom Quantibody arrays. Krunskal-Wallis test was used to compare groups. The diagnostic ability of each biomarker was assessed using chi-square test and ROC analysis. RESULTS: Serum IL-1ß, TNF-α and fibrinogen were significantly higher in CVD patients than those without. Serum fibrinogen displayed a trend of higher concentration in patients with radiographic bone loss (RBL) ≥2 mm (P = 0.08). PICF TNF-α exhibited a significantly higher detection level in the CVD patients that is coincided with the local peri-implant inflammation. In addition, PICF MMP-8 was significantly higher in the RBL ≥2 mm sites than the healthy implants; whereas IL-1ß, IL-8, MMP-8, and TIMP-2 proved to be the significant predictors for peri-implant disease. GCF TNF-α collected from patients with periodontitis was significantly associated with CVD cases. CONCLUSION: The augmented expression of local and systemic pro-inflammatory cytokines found in the current study supports the weak association between the chronic peri-implantitis with increasing severity and CVD.

Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation.

BACKGROUND: RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear. METHODS: To investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach. RESULTS: Here, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion. CONCLUSIONS: Our study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

FOXM1 is required for small cell lung cancer tumorigenesis and associated with poor clinical prognosis.

Small cell lung cancer (SCLC) continues to cause poor clinical outcomes due to limited advances in sustained treatments for rapid cancer cell proliferation and progression. The transcriptional factor Forkhead Box M1 (FOXM1) regulates cell proliferation, tumor initiation, and progression in multiple cancer types. However, its biological function and clinical significance in SCLC remain unestablished. Analysis of the Cancer Cell Line Encyclopedia and SCLC datasets in the present study disclosed significant upregulation of FOXM1 mRNA in SCLC cell lines and tissues. Gene set enrichment analysis (GSEA) revealed that FOXM1 is positively correlated with pathways regulating cell proliferation and DNA damage repair, as evident from sensitization of FOXM1-depleted SCLC cells to chemotherapy. Furthermore, Foxm1 knockout inhibited SCLC formation in the Rb1fl/flTrp53fl/flMycLSL/LSL (RPM) mouse model associated with increased levels of neuroendocrine markers, Ascl1 and Cgrp, and decrease in Yap1. Consistently, FOXM1 depletion in NCI-H1688 SCLC cells reduced migration and enhanced apoptosis and sensitivity to cisplatin and etoposide. SCLC with high FOXM1 expression (N = 30, 57.7%) was significantly correlated with advanced clinical stage, extrathoracic metastases, and decrease in overall survival (OS), compared with the low-FOXM1 group (7.90 vs. 12.46 months). Moreover, the high-FOXM1 group showed shorter progression-free survival after standard chemotherapy, compared with the low-FOXM1 group (3.90 vs. 8.69 months). Our collective findings support the utility of FOXM1 as a prognostic biomarker and potential molecular target for SCLC.

Efficacy of microsurgery and comparison to macrosurgery for gingival recession treatment: a systematic review with meta-analysis.

OBJECTIVES: Microsurgical principles, techniques, and armamentarium have made significant contributions to the periodontal plastic surgery. The present meta-analysis aimed to investigate the overall efficacy of microsurgery on root coverage, and its clinical outcomes when compared to traditional macrosurgery. MATERIAL AND METHODS: Electronic searches on PubMed, Embase, and CINAHL were used to retrieve prospective clinical trials. Primary outcomes were the mean root coverage (mRC) and probability of achieving complete root coverage (cRC), with secondary outcomes as other periodontal parameters and patient-reported outcome measures (PROMs). RESULTS: Nineteen studies were included in the quantitative analysis. Microsurgery was estimated to achieve 83.3% mRC and 69.3% cRC. From a subgroup of 9 comparative studies, it was estimated microsurgery increased mRC by 6.6% (p<0.001) and cRC by 27.9% (p<0.01) compared to macrosurgical control treatments. Operating microscope (OM) yielded a significantly 6.7% higher mRC than the control group (p=0.002), while using loupes showed 6.16% increase in mRC with a borderline significance (p=0.09). OM and loupes-only had a 31.05% (p=0.001) and 25.54% (p=0.001) increases in achieving cRC compared to control, respectively. As for PROMs, microsurgery reduced postoperative pain (p<0.001) and enhanced esthetics (p= 0.05). CONCLUSIONS: Microsurgery significantly improved mean root coverage, probability of achieving complete root coverage, esthetics, and post-surgical recovery. Microsurgery enhances not only subclinical healing but also clinical outcomes, possibly owing to its minimally invasive approach and surgical precision. CLINICAL RELEVANCE: Periodontal plastic microsurgery is minimally invasive, inducing less surgical trauma and ultimately resulting in improved clinical outcomes, patient's satisfaction, and quality of life.

Forkhead Box M1 Transcription Factor Drives Liver Inflammation Linking to Hepatocarcinogenesis in Mice.

BACKGROUND & AIMS: Liver inflammation has been recognized as a hallmark of hepatocarcinogenesis. Although Forkhead Box M1 (FoxM1) is a well-defined oncogenic transcription factor that is overexpressed in hepatocellular carcinoma (HCC), its role in liver inflammation has never been explored. METHODS: We generated hepatocyte-specific FoxM1 conditional transgenic (TG) mice by using the Cre-loxP and Tetracycline (Tet)-on systems to induce FoxM1 expression in a hepatocyte-specific and time-dependent manner. RESULTS: After treatment of Tet-derivatives doxycycline (DOX) to induce FoxM1, TG mice exhibited spontaneous development of hepatocyte death with elevated serum alanine aminotransferase levels and hepatic infiltration of macrophages. The removal of DOX in TG mice completely removed this effect, suggesting that spontaneous inflammation in TG mice occurs in a hepatocyte FoxM1-dependent manner. In addition, liver inflammation in TG mice was associated with increased levels of hepatic and serum chemokine (C-C motif) ligand 2 (CCL2). In vitro transcriptional analysis confirmed that CCL2 is a direct target of FoxM1 in murine hepatocytes. After receiving FoxM1 induction since birth, all TG mice exhibited spontaneous HCC with liver fibrosis at 12 months of age. Hepatic expression of FoxM1 was significantly increased in liver injury models. Finally, pharmacologic inhibition of FoxM1 reduced liver inflammation in models of liver injury. CONCLUSIONS: Hepatocyte FoxM1 acts as a crucial regulator to orchestrate liver inflammation linking to hepatocarcinogenesis. Thus, hepatocyte FoxM1 may be a potential target not only for the treatment of liver injury but also for the prevention toward HCC.

Volumetric facial contour changes of immediately placed implants with and without immediate provisionalization.

BACKGROUND: Whether immediate provisionalization can preserve facial tissue contour remains undetermined. The goal of this 12-month randomized controlled clinical trial was to compare three-dimensional (3D) ridge changes after immediate implant placement with and without immediate provisionalization. METHODS: Forty participants with an unrestorable maxillary anterior or premolar tooth were randomized to receive either a provisional crown (test) or standard healing abutment (control) after immediate implant placement. In each participant, three digital models taken before implant surgery, final crown delivery (4 months), and final follow-up (12 months) were registered to analyze linear deviation in 3D and volume changes of ridge contour at the implant site. RESULTS: The mean value of mid-facial linear 3D spatial resorption ranged from 0.1 to 0.7 mm. Significant difference of linear changes of facial contour was noted over time and not between the groups. Facial volume changes at 12 months remained significantly higher in the control group than in the test group (17.4% versus 11.9%, P = 0.04). CONCLUSIONS: Linear changes of facial soft-tissue resorption at immediately placed implants were independent of immediate provisionalization. However, immediate provisionalization showed better volume preservation at the esthetic concern area (mid-facial margin and 2 to 6 mm above) at the final 12-month follow-up.

Therapeutic inhibition of mTORC2 rescues the behavioral and neurophysiological abnormalities associated with Pten-deficiency.

Dysregulation of the mammalian target of rapamycin (mTOR) signaling, which is mediated by two structurally and functionally distinct complexes, mTORC1 and mTORC2, has been implicated in several neurological disorders1-3. Individuals carrying loss-of-function mutations in the phosphatase and tensin homolog (PTEN) gene, a negative regulator of mTOR signaling, are prone to developing macrocephaly, autism spectrum disorder (ASD), seizures and intellectual disability2,4,5. It is generally believed that the neurological symptoms associated with loss of PTEN and other mTORopathies (for example, mutations in the tuberous sclerosis genes TSC1 or TSC2) are due to hyperactivation of mTORC1-mediated protein synthesis1,2,4,6,7. Using molecular genetics, we unexpectedly found that genetic deletion of mTORC2 (but not mTORC1) activity prolonged lifespan, suppressed seizures, rescued ASD-like behaviors and long-term memory, and normalized metabolic changes in the brain of mice lacking Pten. In a more therapeutically oriented approach, we found that administration of an antisense oligonucleotide (ASO) targeting mTORC2's defining component Rictor specifically inhibits mTORC2 activity and reverses the behavioral and neurophysiological abnormalities in adolescent Pten-deficient mice. Collectively, our findings indicate that mTORC2 is the major driver underlying the neuropathophysiology associated with Pten-deficiency, and its therapeutic reduction could represent a promising and broadly effective translational therapy for neurological disorders where mTOR signaling is dysregulated.

MED28 and forkhead box M1 (FOXM1) mediate matrix metalloproteinase 2 (MMP2)-dependent cellular migration in human nonsmall cell lung cancer (NSCLC) cells.

Non-small-cell lung cancer (NSCLC) accounts for the majority of the lung cancer cases that have become a leading cause of cancer deaths worldwide. Overexpression of transcription factor forkhead box M1 (FOXM1) is involved in the inauspicious development of several types of cancer, including lung tumor aggressiveness. Our laboratory has previously found that MED28, a Mediator subunit for transcriptional activation, modulates cell growth, epithelial-mesenchymal transition, migration, and invasion in human breast cancer cells. The objective of the current study is to investigate the potential role of MED28 and FOXM1 in NSCLC. In addition to A549 and PC9 cells, we also used a doxycycline-inducible system to generate FOXM1-overexpressed A549-DN cells, and we explored the connection of MED28 with FOXM1 and their effect on migration. Herein, we report that the increased expression levels of both MED28 and FOXM1 elevated the expression of matrix metalloproteinase 2 (MMP2), a metastasis marker, which enhanced cell migration and matrigel invasion of NSCLC cells. Furthermore, MED28 interacted with FOXM1, and both exhibited a mutual effect on the expression and subcellular localization. Moreover, MED28 small interfering RNA-mediated MMP2 gene suppression could be attenuated by inducible expression of a constitutively active form of FOXM1, which consequently restored the migration and invasion ability of NSCLC cells. Our data indicate that MED28 interacts with FOXM1, and each affects the expression and localization of the other, and, more importantly, both regulate MMP2-dependent migration and invasion in human lung cancer cells.

Hypoxia induced mitogenic factor (HIMF) triggers angiogenesis by increasing interleukin-18 production in myoblasts.

Inflammatory myopathy is a rare autoimmune muscle disorder. Treatment typically focuses on skeletal muscle weakness or inflammation within muscle, as well as complications of respiratory failure secondary to respiratory muscle weakness. Impaired respiratory muscle function contributes to increased dyspnea and reduced exercise capacity in pulmonary hypertension (PH), a debilitating condition that has few treatment options. The initiation and progression of PH is associated with inflammation and inflammatory cell recruitment and it is established that hypoxia-induced mitogenic factor (HIMF, also known as resistin-like molecule α), activates macrophages in PH. However, the relationship between HIMF and inflammatory myoblasts remains unclear. This study investigated the signaling pathway involved in interleukin-18 (IL-18) expression and its relationship with HIMF in cultured myoblasts. We found that HIMF increased IL-18 production in myoblasts and that secreted IL-18 promoted tube formation of the endothelial progenitor cells. We used the mouse xenograft model and the chick chorioallantoic membrane assay to further explore the role of HIMF in inflammatory myoblasts and angiogenesis in vivo. Thus, our study focused on the mechanism by which HIMF mediates IL-18 expression in myoblasts through angiogenesis in vitro and in vivo. Our findings provide an insight into HIMF functioning in inflammatory myoblasts.

The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and prevents IL-13 and STAT6 signaling in allergen-exposed mice.

Goblet cell metaplasia and excessive mucus secretion associated with asthma, cystic fibrosis, and chronic obstructive pulmonary disease contribute to morbidity and mortality worldwide. We performed a high-throughput screen to identify small molecules targeting a transcriptional network critical for the differentiation of goblet cells in response to allergens. We identified RCM-1, a nontoxic small molecule that inhibited goblet cell metaplasia and excessive mucus production in mice after exposure to allergens. RCM-1 blocked the nuclear localization and increased the proteasomal degradation of Forkhead box M1 (FOXM1), a transcription factor critical for the differentiation of goblet cells from airway progenitor cells. RCM-1 reduced airway resistance, increased lung compliance, and decreased proinflammatory cytokine production in mice exposed to the house dust mite and interleukin-13 (IL-13), which triggers goblet cell metaplasia. In cultured airway epithelial cells and in mice, RCM-1 reduced IL-13 and STAT6 (signal transducer and activator of transcription 6) signaling and prevented the expression of the STAT6 target genes Spdef and Foxa3, which are key transcriptional regulators of goblet cell differentiation. These results suggest that RCM-1 is an inhibitor of goblet cell metaplasia and IL-13 signaling, providing a new therapeutic candidate to treat patients with asthma and other chronic airway diseases.

Peripheral sensory neuron injury contributes to neuropathic pain in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS)-induced neuropathic pain deteriorates quality of life in patients but is often refractory to treatment. In experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, animals develop neuropathy and inflammation-induced tissue acidosis, which suggests the involvement of acid-sensing ion channels (ASICs). Also, peripheral neuropathy is reported in MS patients. However, the involvement of the peripheral nervous system (PNS) in MS neuropathic pain remains elusive. This study investigated the contribution of ASICs and peripheral neuropathy in MS-induced neuropathic pain. Elicited pain levels were as high in Asic1a-/-, Asic2-/- and Asic3-/- mice as wild-type mice even though only Asic1a-/- mice showed reduced EAE disease severity, which indicates that pain in EAE was independent of disease severity. We thus adopted an EAE model without pertussis toxin (EAEnp) to restrain activated immunity in the periphery and evaluate the PNS contribution to pain. Both EAE and EAEnp mice showed similar pain behaviors and peripheral neuropathy in nerve fibers and DRG neurons. Moreover, pregabalin significantly reduced neuropathic pain in both EAE and EAEnp mice. Our findings highlight the essential role of the PNS in neuropathic pain in EAE and pave the way for future development of analgesics without side effects in the CNS.

Characterization of spent nickel-metal hydride batteries and a preliminary economic evaluation of the recovery processes.

UNLABELLED: Valuable metal materials can be recovered from spent nickel-metal hydride (NiMH) batteries. However, little attention has been paid to the metal compositions of individual components of NiMH batteries, although this is important for the selection of the appropriate recycling process. In this study, NiMH batteries were manually disassembled to identify the components and to characterize the metals in each of these. A preliminary economic analysis was also conducted to evaluate the recovery of valuable metals from spent NiMH batteries using thermal melting versus simple mechanical separation. The results of this study show that metallic components account for more than 60% of battery weight. The contents of Ni, Fe, Co, and rare earth elements (REEs) (i.e., valuable metals of interest for recovery) in a single battery were 17.9%, 15.4%, 4.41%, and 17.3%, respectively. Most of the Fe was in the battery components of the steel cathode collector, cathode cap, and anode metal grid, while Ni (>90%) and Co (>90%) were mainly in the electrode active materials (anode and cathode metal powders). About 1.88 g of REEs (Ce, La, and Y) could be obtained from one spent NiMH battery. The estimated profits from recovering valuable metals from spent NiMH batteries by using thermal melting and mechanical processes are 2,329 and 2,531 USD/ton, respectively, when including a subsidy of 1,710 USD/ton. The findings of this study are very useful for further research related to technical and economic evaluations of the recovery of valuable metals from spent NiMH batteries. IMPLICATIONS: The spent nickel-metal hydride (NiMH) batteries were manually disassembled and their components were identified. The metals account for more than 60% of battery weight, when Ni, Fe, Co, and rare earth elements (REEs) were 17.9%, 15.4%, 4.41%, and 17.3%, respectively, in a single battery. The estimated profits of recovering valuable metals from NiMH batteries by using thermal melting and mechanical processing are 2,329 and 2,531 USD/ton, respectively, when including a subsidy of 1,710 USD/ton. These findings are very useful to develop or select the recovery methods of valuable metals from spent NiMH batteries.

An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.

Foxm1 transcription factor is critical for proliferation and differentiation of Clara cells during development of conducting airways.

Respiratory epithelial cells are derived from cell progenitors in the foregut endoderm that subsequently differentiate into the distinct cell types lining the conducting and alveolar regions of the lung. To identify transcriptional mechanisms regulating differentiation and maintenance of respiratory epithelial cells, we conditionally deleted Foxm1 transcription factor from the conducting airways of the developing mouse lung. Conditional deletion of Foxm1 from Clara cells, controlled by the Scgb1a1 promoter, dramatically altered airway structure and caused peribronchial fibrosis, resulting in airway hyperreactivity in adult mice. Deletion of Foxm1 inhibited proliferation of Clara cells and disrupted the normal patterning of epithelial cell differentiation in the bronchioles, causing squamous and goblet cell metaplasia, and the loss of Clara and ciliated cells. Surprisingly, conducting airways of Foxm1-deficient mice contained highly differentiated cuboidal type II epithelial cells that are normally restricted to the alveoli. Lineage tracing studies showed that the ectopic alveolar type II cells in Foxm1-deficient airways were derived from Clara cells. Deletion of Foxm1 inhibited Sox2 and Scgb1a1, both of which are critical for differentiation and function of Clara cells. In co-transfection experiments, Foxm1 directly bound to and induced transcriptional activity of Scgb1a1 and Sox2 promoters. Foxm1 is required for differentiation and maintenance of epithelial cells lining conducting airways.

Foxm1 mediates cross talk between Kras/mitogen-activated protein kinase and canonical Wnt pathways during development of respiratory epithelium.

While Kras/mitogen-activated protein kinase (MAPK) and canonical Wnt/ß-catenin are critical for lung morphogenesis, mechanisms integrating these important signaling pathways during lung development are unknown. Herein, we demonstrate that the Foxm1 transcription factor is a key downstream target of activated Kras(G12D). Deletion of Foxm1 from respiratory epithelial cells during lung formation prevented structural abnormalities caused by activated Kras(G12D). Kras/Foxm1 signaling inhibited the activity of canonical Wnt signaling in the developing lung in vivo. Foxm1 decreased T-cell factor (TCF) transcriptional activity induced by activated ß-catenin in vitro. Depletion of Foxm1 by short interfering RNA (siRNA) increased nuclear localization of ß-catenin, increased expression of ß-catenin target genes, and decreased mRNA and protein levels of the ß-catenin inhibitor Axin2. Axin2 mRNA was reduced in distal lung epithelium of Foxm1-deficient mice. Foxm1 directly bound to and increased transcriptional activity of the Axin2 promoter region. Foxm1 is required for Kras signaling in distal lung epithelium and provides a mechanism integrating Kras and canonical Wnt/ß-catenin signaling during lung development.

Expression of Foxm1 transcription factor in cardiomyocytes is required for myocardial development.

Forkhead Box M1 (Foxm1) is a transcription factor essential for organ morphogenesis and development of various cancers. Although complete deletion of Foxm1 in Foxm1(-/-) mice caused embryonic lethality due to severe abnormalities in multiple organ systems, requirements for Foxm1 in cardiomyocytes remain to be determined. This study was designed to elucidate the cardiomyocyte-autonomous role of Foxm1 signaling in heart development. We generated a new mouse model in which Foxm1 was specifically deleted from cardiomyocytes (Nkx2.5-Cre/Foxm1(fl/f) mice). Deletion of Foxm1 from cardiomyocytes was sufficient to disrupt heart morphogenesis and induce embryonic lethality in late gestation. Nkx2.5-Cre/Foxm1(fl/fl) hearts were dilated with thinning of the ventricular walls and interventricular septum, as well as disorganization of the myocardium which culminated in cardiac fibrosis and decreased capillary density. Cardiomyocyte proliferation was diminished in Nkx2.5-Cre/Foxm1(fl/fl) hearts owing to altered expression of multiple cell cycle regulatory genes, such as Cdc25B, Cyclin B(1), Plk-1, nMyc and p21(cip1). In addition, Foxm1 deficient hearts displayed reduced expression of CaMKIIδ, Hey2 and myocardin, which are critical mediators of cardiac function and myocardial growth. Our results indicate that Foxm1 expression in cardiomyocytes is critical for proper heart development and required for cardiomyocyte proliferation and myocardial growth.