Sessile alveolar macrophage connexin-43 determines mechano-immunity in the lung.

Although lung immunity is pathogen induced, the immunity can also be induced by mechanical distortion of the lung. The causal basis of the lung's mechanosensitive immunity remains unclear. Here, through live optical imaging of mouse lungs, we show that alveolar stretch due to hyperinflation induced prolonged cytosolic Ca2+ increases in sessile alveolar macrophages (AMs). Knockout studies revealed that the Ca2+ increases resulted from Ca2+ diffusion from the alveolar epithelium to sessile AMs through connexin 43 (Cx43)-containing gap junctions. Lung inflammation and injury in mice exposed to injurious mechanical ventilation were inhibited by AM-specific Cx43 knockout, or AM-specific delivery of a calcium inhibitor. We conclude, Cx43 gap junctions and calcium mobilization in sessile AMs determine the lung's mechanosensitive immunity, providing a therapeutic strategy against hyperinflation-induced lung injury.

Retinoids stored locally in the lung are required to attenuate the severity of acute lung injury in male mice.

Retinoids are potent transcriptional regulators that act in regulating cell proliferation, differentiation, and other cellular processes. We carried out studies in male mice to establish the importance of local cellular retinoid stores within the lung alveolus for maintaining its health in the face of an acute inflammatory challenge induced by intranasal instillation of lipopolysaccharide. We also undertook single cell RNA sequencing and bioinformatic analyses to identify roles for different alveolar cell populations involved in mediating these retinoid-dependent responses. Here we show that local retinoid stores and uncompromised metabolism and signaling within the lung are required to lessen the severity of an acute inflammatory challenge. Unexpectedly, our data also establish that alveolar cells other than lipofibroblasts, specifically microvascular endothelial and alveolar epithelial cells, are able to take up lipoprotein-transported retinoid and to accumulate cellular retinoid stores that are directly used to respond to an acute inflammatory challenge.

The mitochondrial calcium uniporter of pulmonary type 2 cells determines severity of acute lung injury.

Acute Lung Injury (ALI) due to inhaled pathogens causes high mortality. Underlying mechanisms are inadequately understood. Here, by optical imaging of live mouse lungs we show that a key mechanism is the viability of cytosolic Ca2+ buffering by the mitochondrial Ca2+ uniporter (MCU) in the lung's surfactant-secreting, alveolar type 2 cells (AT2). The buffering increased mitochondrial Ca2+ and induced surfactant secretion in wild-type mice, but not in mice with AT2-specific MCU knockout. In the knockout mice, ALI due to intranasal LPS instillation caused severe pulmonary edema and mortality, which were mitigated by surfactant replenishment prior to LPS instillation, indicating surfactant's protective effect against alveolar edema. In wild-type mice, intranasal LPS, or Pseudomonas aeruginosa decreased AT2 MCU. Loss of MCU abrogated buffering. The resulting mortality was reduced by spontaneous recovery of MCU expression, or by MCU replenishment. Enhancement of AT2 mitochondrial buffering, hence endogenous surfactant secretion, through MCU replenishment might be a therapy against ALI.

Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway.

Multiple beneficial cardiovascular effects of HDL depend on sphingosine-1-phosphate (S1P). S1P associates with HDL by binding to apolipoprotein M (ApoM). Insulin resistance is a major driver of dyslipidemia and cardiovascular risk. However, the mechanisms linking alterations in insulin signaling with plasma lipoprotein metabolism are incompletely understood. The insulin-repressible FoxO transcription factors mediate key effects of hepatic insulin action on glucose and lipoprotein metabolism. This work tested whether hepatic insulin signaling regulates HDL-S1P and aimed to identify the underlying molecular mechanisms. We report that insulin-resistant, nondiabetic individuals had decreased HDL-S1P levels, but no change in total plasma S1P. This also occurred in insulin-resistant db/db mice, which had low ApoM and a specific reduction of S1P in the HDL fraction, with no change in total plasma S1P levels. Using mice lacking hepatic FoxOs (L-FoxO1,3,4), we found that hepatic FoxOs were required for ApoM expression. Total plasma S1P levels were similar to those in controls, but S1P was nearly absent from HDL and was instead increased in the lipoprotein-depleted plasma fraction. This phenotype was restored to normal by rescuing ApoM in L-FoxO1,3,4 mice. Our findings show that insulin resistance in humans and mice is associated with decreased HDL-associated S1P. Our study shows that hepatic FoxO transcription factors are regulators of the ApoM/S1P pathway.

Modulation of the NLRP3 inflammasome by Sars-CoV-2 Envelope protein.

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.

Actin fence therapy with exogenous V12Rac1 protects against acute lung injury.

High mortality in acute lung injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF-α receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-α-induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss, and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally (i.n.) as a noncovalent construct with a cell-permeable peptide, enhanced alveolar filamentous actin (F-actin) and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from i.n. instillation of LPS or of Pseudomonas aeruginosa. We propose a potentially new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, and therefore blocking ALI.

The Mitochondrial Calcium Uniporter of Pulmonary Type 2 Cells Determines Severity of ARDS.

Acute lung immunity to inhaled pathogens elicits defensive pneumonitis that may convert to the Acute Respiratory Distress Syndrome (ARDS), causing high mortality. Mechanisms underlying the conversion are not understood, but are of intense interest because of the ARDS-induced mortality in the ongoing Covid-19 pandemic. Here, by optical imaging of live lungs we show that key to the lethality is the functional status of mitochondrial Ca2+ buffering across the mitochondrial Ca2+ uniporter (MCU) in the alveolar type 2 cells (AT2), which protect alveolar stability. In mice subjected to ARDS by airway exposure to lipopolysaccharide (LPS), or to Pseudomonas aeruginosa, there was marked loss of MCU expression in AT2. The ability of mice to survive ARDS depended on the extent to which the MCU expression recovered, indicating that the viability of Ca2+ buffering by AT2 mitochondria critically determines ARDS severity. Mitochondrial transfer to enhance AT2 MCU expression might protect against ARDS.

Endothelial mitochondria determine rapid barrier failure in chemical lung injury.

Acid aspiration, which can result from several etiologies, including postoperative complications, leads to direct contact of concentrated hydrochloric acid (HCl) with the alveolar epithelium. As a result, rapid endothelial activation induces alveolar inflammation, leading to life-threatening pulmonary edema. Because mechanisms underlying the rapid endothelial activation are not understood, here we determined responses in real time through optical imaging of alveoli of live mouse lungs. By alveolar micropuncture, we microinfused concentrated HCl in the alveolar lumen. As expected, acid contact with the epithelium caused rapid, but transient, apical injury. However, there was no concomitant membrane injury to the endothelium. Nevertheless, H2O2-mediated epithelial-endothelial paracrine signaling induced endothelial barrier failure, as detected by microvascular dextran leakage and lung water quantification. Remarkably, endothelial mitochondria regulated the barrier failure by activating uncoupling protein 2 (UCP2), thereby inducing transient mitochondrial depolarization that led to cofilin-induced actin depolymerization. Knockdown, or endothelium-targeted deletion of UCP2 expression, blocked these responses, including pulmonary edema. To our knowledge, these findings are the first to mechanistically implicate endothelial mitochondria in acid-induced barrier deterioration and pulmonary edema. We suggest endothelial UCP2 may be a therapeutic target for acid-induced acute lung injury.

Hypercapnia attenuates ventilator-induced lung injury via a disintegrin and metalloprotease-17.

Hypercapnic acidosis, common in mechanically ventilated patients, has been reported to exert both beneficial and harmful effects in models of lung injury. Understanding its effects at the molecular level may provide insight into mechanisms of injury and protection. The aim of this study was to establish the effects of hypercapnic acidosis on mitogen­activated protein kinase (MAPK) activation, and determine the relevant signalling pathways. p44/42 MAPK activation in a murine model of ventilator­induced lung injury (VILI) correlated with injury and was reduced in hypercapnia. When cultured rat alveolar epithelial cells were subjected to cyclic stretch, activation of p44/42 MAPK was dependent on epidermal growth factor receptor (EGFR) activity and on shedding of EGFR ligands; exposure to 12% CO2 without additional buffering blocked ligand shedding, as well as EGFR and p44/42 MAPK activation. The EGFR ligands are known substrates of the matrix metalloprotease ADAM17, suggesting stretch activates and hypercapnic acidosis blocks stretch­mediated activation of ADAM17. This was corroborated in the isolated perfused mouse lung, where elevated CO2 also inhibited stretch­activated shedding of the ADAM17 substrate TNFR1 from airway epithelial cells. Finally, in vivo confirmation was obtained in a two­hit murine model of VILI where pharmacological inhibition of ADAM17 reduced both injury and p44/42 MAPK activation. Thus, ADAM17 is an important proximal mediator of VILI; its inhibition is one mechanism of hypercapnic protection and may be a target for clinical therapy.

Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity.

The tissue-resident macrophages of barrier organs constitute the first line of defence against pathogens at the systemic interface with the ambient environment. In the lung, resident alveolar macrophages (AMs) provide a sentinel function against inhaled pathogens. Bacterial constituents ligate Toll-like receptors (TLRs) on AMs, causing AMs to secrete proinflammatory cytokines that activate alveolar epithelial receptors, leading to recruitment of neutrophils that engulf pathogens. Because the AM-induced response could itself cause tissue injury, it is unclear how AMs modulate the response to prevent injury. Here, using real-time alveolar imaging in situ, we show that a subset of AMs attached to the alveolar wall form connexin 43 (Cx43)-containing gap junction channels with the epithelium. During lipopolysaccharide-induced inflammation, the AMs remained sessile and attached to the alveoli, and they established intercommunication through synchronized Ca(2+) waves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involving Ca(2+)-dependent activation of Akt, because AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the bronchoalveolar lavage. A picture emerges of a novel immunomodulatory process in which a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation.

F-actin scaffold stabilizes lamellar bodies during surfactant secretion.

Alveolar type 2 (AT2) cells secrete surfactant that forms a protective layer on the lung's alveolar epithelium. Vesicles called lamellar bodies (LBs) store surfactant. Failure of surfactant secretion, which causes severe lung disease, relates to the manner in which LBs undergo exocytosis during the secretion. However, the dynamics of LBs during the secretion process are not known in intact alveoli. Here, we addressed this question through real-time confocal microscopy of single AT2 cells in live alveoli of mouse lungs. Using a combination of phospholipid and aqueous fluorophores that localize to LBs, we induced surfactant secretion by transiently hyperinflating the lung, and we quantified the secretion in terms of loss of bulk LB fluorescence. In addition, we quantified inter-LB phospholipid flow through determinations of fluorescence recovery after photobleaching. Furthermore, we determined the role of F-actin in surfactant secretion through expression of the fluorescent F-actin probe Lifeact. Our findings indicate that, in AT2 cells in situ, LBs are held in an F-actin scaffold. Although F-actin transiently decreases during surfactant secretion, the LBs remain stationary, forming a chain of vesicles connected by intervesicular channels that convey surfactant to the secretion site on the plasma membrane. This is the first instance of a secretory process in which the secretory vesicles are immobile, but form a conduit for the secretory material.

Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation.

To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca(2+) from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca(2+) release-activated Ca(2+) (CRAC) channels. This increase in intracellular Ca(2+) induces Ca(2+)/calmodulin-dependent kinase kinase ß (CaMKKß)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca(2+) concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca(2+) signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.

Alpha1-AMP-activated protein kinase regulates hypoxia-induced Na,K-ATPase endocytosis via direct phosphorylation of protein kinase C zeta.

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase C zeta (PKC zeta)-mediated phosphorylation of the Na,K-ATPase alpha subunit. Here, we report that hypoxia leads to the phosphorylation of 5'-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK alpha subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient rho(0)-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKC zeta translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK alpha phosphorylates PKC zeta on residue Thr410 within the PKC zeta activation loop. Importantly, the activation of AMPK alpha was necessary for hypoxia-induced AMPK-PKC zeta binding in alveolar epithelial cells. The overexpression of T410A mutant PKC zeta prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKC zeta Thr410 phosphorylation is essential for this process. PKC zeta activation by AMPK is isoform specific, as small interfering RNA targeting the alpha1 but not the alpha2 catalytic subunit prevented PKC zeta activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK alpha1 isoform, which binds and directly phosphorylates PKC zeta at Thr410, thereby promoting Na,K-ATPase endocytosis.

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.

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.

The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer.

The proliferation-specific Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) regulates expression of cell cycle genes essential for progression into DNA replication and mitosis. Expression of Foxm1 is found in a variety of distinct human cancers including hepatocellular carcinomas, intrahepatic cholangiocarcinomas, basal cell carcinomas, ductal breast carcinomas, and anaplastic astrocytomas and glioblastomas. In this study, we show that human Foxm1 protein is abundantly expressed in highly proliferative human non-small cell lung cancers (NSCLC) as well as in mouse lung tumors induced by urethane. To determine the role of Foxm1 during the development of mouse lung tumors, we used IFN-inducible Mx-Cre recombinase transgene to delete mouse Foxm1 fl/fl-targeted allele before inducing lung tumors with urethane. We show that Mx-Cre Foxm1-/- mice exhibit diminished proliferation of lung tumor cells causing a significant reduction in number and size of lung adenomas. Transient transfection experiments with A549 lung adenocarcinoma cells show that depletion of Foxm1 levels by short interfering RNA caused diminished DNA replication and mitosis and reduced anchorage-independent growth of cell colonies on soft agar. Foxm1-depleted A549 cells exhibit reduced expression of cell cycle-promoting cyclin A2 and cyclin B1 genes. These data show that Foxm1 stimulates the proliferation of tumor cells during progression of NSCLC.

The forkhead box m1 transcription factor is essential for embryonic development of pulmonary vasculature.

Transgenic and gene knock-out studies demonstrated that the mouse Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is essential for hepatocyte entry into mitosis during liver development, regeneration, and liver cancer. Targeted deletion of Foxm1 gene in mice produces an embryonic lethal phenotype due to severe abnormalities in the development of liver and heart. In this study, we show for the first time that Foxm1(-/-) lungs exhibit severe hypertrophy of arteriolar smooth muscle cells and defects in the formation of peripheral pulmonary capillaries as evidenced by significant reduction in platelet endothelial cell adhesion molecule 1 staining of the distal lung. Consistent with these findings, significant reduction in proliferation of the embryonic Foxm1(-/-) lung mesenchyme was found, yet proliferation levels were normal in the Foxm1-deficient epithelial cells. Severe abnormalities of the lung vasculature in Foxm1(-/-) embryos were associated with diminished expression of the transforming growth factor beta receptor II, a disintegrin and metalloprotease domain 17 (ADAM-17), vascular endothelial growth factor receptors, Polo-like kinase 1, Aurora B kinase, laminin alpha4 (Lama4), and the Forkhead Box f1 transcription factor. Cotransfection studies demonstrated that Foxm1 stimulates transcription of the Lama4 promoter, and this stimulation requires the Foxm1 binding sites located between -1174 and -1145 bp of the mouse Lama4 promoter. In summary, development of mouse lungs depends on the Foxm1 transcription factor, which regulates expression of genes essential for mesenchyme proliferation, extracellular matrix remodeling, and vasculogenesis.

The mouse Forkhead Box m1 transcription factor is essential for hepatoblast mitosis and development of intrahepatic bile ducts and vessels during liver morphogenesis.

Conditional deletion of the mouse Forkhead Box (Fox) m1b targeted allele in adult hepatocytes (Foxm1, previously called HFH-11B, Trident, Win, or MPP2) demonstrated that the Foxm1b transcription factor is essential for hepatocyte mitosis during liver regeneration. To determine the role of Foxm1b in liver development, we have generated Foxm1b -/- mice that deleted the Foxm1b exons encoding the winged helix DNA binding and transcriptional activation domains. Here, we show that all of the Foxm1b -/- embryos died in utero by 18.5 days postcoitum (dpc). Embryonic Foxm1b -/- livers displayed a 75% reduction in the number of hepatoblasts, resulting from diminished DNA replication and a failure to enter mitosis causing a polyploid phenotype. Reduced hepatoblast mitosis was associated with decreased protein levels of the Polo-like kinase 1 and Aurora B kinase, which phosphorylate regulatory proteins essential for orchestrating mitosis and cytokinesis. Diminished proliferation of Foxm1b -/- hepatoblasts contributed to abnormal liver development with significant reduction in the number of large hepatic veins compared to embryonic wild-type (WT) liver. Furthermore, embryonic Foxm1b -/- livers did not develop intrahepatic bile ducts, and these presumptive biliary hepatoblasts failed to express either biliary cytokeratins or nuclear levels of hepatocyte nuclear factor 1beta. These results suggest that Foxm1b is critical for hepatoblast precursor cells to differentiate toward biliary epithelial cell lineage. Finally, we used a hepatoblast-specific Cre recombinase transgene to mediate deletion of the Foxm1b fl/fl allele in the developing liver, and these embryos died in utero and exhibited diminished hepatoblast proliferation with similar abnormalities in liver morphogenesis, suggesting that the defect in liver development contributed to embryonic lethality.

Foxf1 haploinsufficiency reduces Notch-2 signaling during mouse lung development.

The forkhead box (Fox) f1 transcription factor is expressed in the mouse splanchnic (visceral) mesoderm, which contributes to development of the liver, gallbladder, lung, and intestinal tract. Pulmonary hemorrhage and peripheral microvascular defects were found in approximately half of the newborn Foxf1(+/-) mice, which expressed low levels of lung Foxf1 mRNA [low-Foxf1(+/-) mice]. Microvascular development was normal in the surviving newborn high-Foxf1(+/-) mice, which compensated for pulmonary Foxf1 haploinsufficiency and expressed wild-type Foxf1 levels. To identify expression of genes regulated by Foxf1, we used Affymetrix microarrays to determine embryonic lung RNAs influenced by Foxf1 haploinsufficiency. Embryonic Foxf1(+/-) lungs exhibited diminished expression of hepatocyte growth factor receptor c-Met, myosin VI, the transcription factors SP-3, BMI-1, ATF-2, and glucocorticoid receptor, and cell cycle inhibitors p53, p21(Cip1), retinoblastoma, and p107. Furthermore, Notch-2 signaling was decreased in embryonic Foxf1(+/-) lungs, as evidenced by significantly reduced levels of the Notch-2 receptor and the Notch-2 downstream target hairy enhancer of split-1. The severity of the Notch-2-signaling defect in 18-day postcoitus Foxf1(+/-) lungs correlated with Foxf1 mRNA levels. Disruption of pulmonary Notch-2 signaling continued in newborn low-Foxf1(+/-) mice, which died of lung hemorrhage and failed to compensate for Foxf1 haploinsufficiency. In contrast, in newborn high-Foxf1(+/-) lungs, Notch-2 signaling was restored to the level found in wild-type mice, which was associated with normal microvascular formation and survival. Foxf1 haploinsufficiency disrupted pulmonary expression of genes in the Notch-2-signaling pathway and resulted in abnormal development of lung microvasculature.

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.