Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the ␣-and ␤-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both ␣-and ␤-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Alveolar epithelial cells are directly exposed to acute and chronic fluctuations in alveolar oxygen tension. Previously, we found that the oxygen-binding protein hemoglobin is expressed in alveolar Type II cells (ATII). Here, we report that ATII cells also express a number of highly specific transcription factors and other genes normally associated with hemoglobin biosynthesis in erythroid precursors. Because hypoxia-inducible factors (HIFs) were shown to play a role in hypoxia-induced changes in ATII homeostasis, we hypothesized that the hypoxia-induced increase in intracellular HIF exerts a concomitant effect on ATII hemoglobin expression. Treatment of cells from the ATII-like immortalized mouse lung epithelial cell line-15 (MLE-15) with hypoxia for 20 hours resulted in dramatic increases in cellular levels of HIF-2a protein and parallel significant increases in hemoglobin messenger RNA (mRNA) and protein expression, as compared with that of control cells cultured in normoxia. Significant increases in the mRNA of globin-associated transcription factors were also observed, and RNA interference (RNAi) experiments demonstrated that the expression of hemoglobin is at least partially dependent on the cellular levels of globin-associated transcription factor isoform 1 (GATA-1). Conversely, levels of prosurfactant proteins B and C significantly decreased in the same cells after exposure to hypoxia. The treatment of MLE-15 cells cultured in normoxia with prolyl 4-hydroxylase inhibitors, which mimic the effects of hypoxia, resulted in increases of hemoglobin and decreases of surfactant proteins. Taken together, these results suggest a relationship between hypoxia, HIFs, and the expression of hemoglobin, and imply that hemoglobin may be involved in the oxygen-sensing pathway in alveolar epithelial cells.Keywords: hemoglobin; alveolar epithelial cells; hypoxia; hypoxiainducible factor Pulmonary hypoxia occurs under both physiologic and pathologic conditions. In fact, a hypoxic environment is necessary for proper embryonic lung development, by promoting the formation of microvasculature and epithelial branching morphogenesis (the average fetal blood O 2 fraction is z2-5%) (1-3). However, postnatal decreases in alveolar oxygen tension as a result of pulmonary disease disrupt alveolar homeostasis. High-altitude ascent, pathologic conditions resulting in inadequate respiration, pulmonary edema after acute lung injury, or congestive heart failure may all result in decreased oxygen tension. Alveolar Type II (ATII) cells represent approximately two thirds of epithelial cell numbers, and are of special clinical interest because of their role in the production, secretion, and recycling of pulmonary surfactant (4). In addition, ATII cells differentiate into Type I (ATI ) cells upon epithelial injury, and also act to clear fluid from the alveolar space. Although numerous studies evaluated the effects of hypoxia on the pulmonary endothelium, few sought to identify hypoxia-regulated genes in alveolar epithelial cells.Our previous stu...

Patients with mutations in the pulmonary surfactant protein C (SP-C) gene develop interstitial lung disease and pulmonary exacerbations associated with viral infections including respiratory syncytial virus (RSV). Pulmonary infection with RSV caused more severe interstitial thickening, air space consolidation, and goblet cell hyperplasia in SP-C-deficient ( Sftpc−/−) mice compared with SP-C replete mice. The RSV-induced pathology resolved more slowly in Sftpc−/−mice with lung inflammation persistent up to 30 days postinfection. Polymorphonuclear leukocyte and macrophage counts were increased in the bronchoalveolar lavage (BAL) fluid of Sftpc−/−mice. Viral titers and viral F and G protein mRNA were significantly increased in both Sftpc−/−and heterozygous Sftpc+/−mice compared with controls. Expression of Toll-like receptor 3 (TLR3) mRNA was increased in the lungs of Sftpc−/−mice relative to Sftpc+/+mice before and after RSV infection. Consistent with the increased TLR3 expression, BAL inflammatory cells were increased in the Sftpc−/−mice after exposure to a TLR3-specific ligand, poly(I:C). Preparations of purified SP-C and synthetic phospholipids blocked poly(I:C)-induced TLR3 signaling in vitro. SP-C deficiency increases the severity of RSV-induced pulmonary inflammation through regulation of TLR3 signaling.

Background Polymorphic alleles of the vitamin D (vitD)-binding protein (VDBP) gene are associated with discriminatory differences in circulating concentrations of 25-hydroxyvitamin D (25-D), the indicator of vitD status (sufficiency defined by the Endocrine Society as ≥75 nmol/L). Within a diverse group of children, we hypothesized that reaching recommended daily allowance (RDA) of vitD intake would have differential impact on vitD status depending on VDBP variability. Methods VDBP alleles (Gc1S, Gc1F, Gc2) in 123 children (1–4 annual visits/child; ages 1–8 years) were compared for relationships with serum 25-D concentrations and daily vitD intake. Results In African-American children, reaching the vitD RDA was associated with significantly higher mean serum 25-D concentrations for the 20% carrying the VDBP 1S allele than for the large majority without this allele (77 vs. 61 nmol/L 25-D; p = 0.038). Children with the Gc1S/1S homozygous genotype (30% Caucasians, 24% Hispanics, 2% African-Americans) who met RDA had 51% (39 nmol/L) greater mean serum 25-D than those below RDA ( p < 0.0001). Conclusions VDBP genetic variability was a significant factor affecting childhood vitD status when following RDA guidelines. This study may inform public health policy of uniformity in recommended childhood vitD dosage, especially regarding racially/ethnically associated disparities.

Lottes RG, Newton DA, Spyropoulos DD, Baatz JE. Lactate as substrate for mitochondrial respiration in alveolar epithelial type II cells. Am J Physiol Lung Cell Mol Physiol 308: L953-L961, 2015. First published March 6, 2015 doi:10.1152/ajplung.00335.2014.-Because of the many energy-demanding functions they perform and their physical location in the lung, alveolar epithelial type II (ATII) cells have a rapid cellular metabolism and the potential to influence substrate availability and bioenergetics both locally in the lung and throughout the body. A thorough understanding of ATII cell metabolic function in the healthy lung is necessary for determining how metabolic changes may contribute to pulmonary disease pathogenesis; however, lung metabolism is poorly understood at the cellular level. Here, we examine lactate utilization by primary ATII cells and the ATII model cell line, MLE-15, and link lactate consumption directly to mitochondrial ATP generation. ATII cells cultured in lactate undergo mitochondrial respiration at near-maximal levels, two times the rates of those grown in glucose, and oxygen consumption under these conditions is directly linked to mitochondrial ATP generation. When both lactate and glucose are available as metabolic substrate, the presence of lactate alters glucose metabolism in ATII to favor reduced glycolytic function in a dose-dependent manner, suggesting that lactate is used in addition to glucose when both substrates are available. Lactate use by ATII mitochondria is dependent on monocarboxylate transporter (MCT)-mediated import, and ATII cells express MCT1, the isoform that mediates lactate import by cells in other lactate-consuming tissues. The balance of lactate production and consumption may play an important role in the maintenance of healthy lung homeostasis, whereas disruption of lactate consumption by factors that impair mitochondrial metabolism, such as hypoxia, may contribute to lactic acid build-up in disease. mitochondrial function; metabolism; hypoxia FROM A METABOLIC perspective, the lung is a unique physiological environment. The cells that compose the alveolar epithelium form the barrier between external air and the pulmonary vasculature in the best-oxygenated environment in the body. While terminally differentiated alveolar epithelial type I (ATI) cells form the passive surface across which gas exchange occurs, alveolar epithelial type II (ATII) cells perform a variety of energetically costly functions, including pulmonary surfactant production (16), fluid transport and homeostasis (27), immune functions (6, 23), and progenitor roles for self-renewal and transdifferentiation to repopulate ATI cells (5, 19). Additionally, the lung is the only organ apart from the heart itself that receives the entire cardiac output upon every passage through the body. Metabolic activity in the cells that compose the alveolar epithelium could potentially influence substrate availability, energy production, and redox balance locally and throughout the whole body (20, 32). While the lung...