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 mitogen‐activated protein kinase (MAPK) activation, and determine the relevant signalling pathways. p44/42 MAPK activation in a murine model of ventilator‐induced 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 stretch‐mediated activation of ADAM17. This was corroborated in the isolated perfused mouse lung, where elevated CO2 also inhibited stretch‐activated shedding of the ADAM17 substrate TNFR1 from airway epithelial cells. Finally, in vivo confirmation was obtained in a two‐hit 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.

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.

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.

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.

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.

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.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging.

Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro, colonic organoids cultured from GPR56-/- mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro, and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo, deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.

The incidence of esophageal adenocarcinoma (EAC) is increasing, but factors contributing to malignant progression of its precursor lesion, Barrett's esophagus (BE), have not been defined. Hypergastrinemia caused by long-term use of proton pump inhibitors (PPIs), has been suggested as one possible risk factor. The gastrin receptor, CCK2R, is expressed in the cardia and upregulated in BE, suggesting the involvement of the gastrin-CCK2R pathway in progression. In the L2-IL-1β mouse model, Barrett's-like esophagus arises from the gastric cardia. Therefore, we aimed to analyze the effect of hypergastrinemia on CCK2R+ progenitor cells in L2-IL-1β mice.

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b(+)Ly6G(+) immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b(+)Ly6G(+) cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b(+)Ly6G(+) IMCs in early cancer development.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

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.