In the present study, we explore the biological function of ET-2 using knockout mouse models. Mice with constitutive or systemically-induced deletion of ET-2 exhibited defective energy homeostasis, thermoregulation, and lung morphology and function. Our comprehensive analyses, including the examination of tissue-specific knockouts, show that intestinal and neuronal ET-2 is not fully accountable for the regulation of energy homeostasis and body temperature, respectively. The dramatic effects of ET-2 deficiency in the lung are newly discovered and the lung may therefore be an important site for critical ET-2 action. In the lung, Et2
mRNA is expressed in epithelial cells and transiently increased soon after birth. Temporary induction of Et2
mRNA by pathological or physiological stimuli is also observed in the photoreceptor cells of the retina (15
) and the granulose cells of the ovary (17
). The analysis of lung-specific Et2
-deficient mice will help to determine whether ET-2 expression in the lung is critical for the growth regulation and survival of postnatal mice. Lung epithelium is composed of several specific cell types such as squamous alveolar (type I) and great alveolar (type II) cells in the alveolar epithelium, and ciliated cells and Clara cells in the respiratory bronchioles. Cell type–specific gene promoters expressing Cre are available for each lung epithelial cell population: surfactant protein C (SP-C) for type II cells, Clara cell secretory protein (CCSP) for Clara cells, and forkhead box J1 (Foxj1) for ciliated cells (32
). Future studies will be needed to determine the specific epithelial cell population responsible for ET-2 expression and thus the most appropriate promoter for removal of ET-2 in the lung.
In this report, internal starvation, severe hypothermia, and lung dysfunction caused by ET-2 deficiency are proposed as possible independent underlying mechanisms explaining the growth retardation and early lethality of Et2
-null mice. Nonetheless, it is highly possible that these phenotypes are not independent, since metabolic rate, body temperature, and breathing regulation are physiologically interrelated. First, starvation might be the primary abnormality caused by the absence of ET-2. Under the stressful environment imposed by reduced food availability and a low ambient temperature, small mammals, including mice, can enter torpor to conserve energy by dropping their body temperature to near-ambient temperature levels (34
). Since nutritional availability is an important determinant of normal lung development, chronic nutrient restriction may induce structural and functional alterations in the lung (35
). The lungs of rats starved soon after birth exhibit impaired alveolar development and enlarged air spaces consistent with the phenotype of Et2
-null mice (36
). Starvation or calorie restriction in adult animals including humans can also lead to structural and functional changes in the lung (37
). Although the lung abnormalities were less marked than what was seen in the Et2
-null mice, these studies raise the possibility that hypothermia and lung dysfunction might be a secondary effect of internal starvation due to ET-2 deficiency.
Another possibility is that lung dysfunction is the primary abnormality underlying the phenotype of Et2
-deficient mice. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. The core fucosylation (α1, 6-fucosylation) of TGF-β1 receptors is crucial for the prevention of developmental and progressive/destructive emphysema. Pdgfa
and α1, 6-fucosyltransferase–null (Fut8
-null) mice display air-space enlargement with the failure of alveolar septation in the lung accompanied by postnatal growth retardation and early lethality (38
). Malnutrition and weight loss are common problems in chronic obstructive pulmonary disease (COPD) (40
), and significant loss of body weight is also observed in mouse models exposed to hypoxia (41
). Many studies have reported that hypoxia evokes a regulated decrease in body temperature (i.e., anapyrexia) as a compensatory response to decreased oxygen consumption (42
). Chronic hypercapnia can impair thermoregulation by increasing sweating and reducing shivering in humans (43
). Thus, it is quite reasonable to hypothesize that in Et2
-null mice, an internally starved state and hypothermia are the consequence, and not the cause, of severe lung dysfunction and respiratory failure.
Whether or not the profound hypothermia of Et2
-null mice is a byproduct of internal starvation and lung dysfunction, it is still an important contributor to the phenotype. Since Et2
-null mice reared in warm conditions lived longer than their null littermates housed at room temperature (Figure F), abnormal thermoregulation may play a critical role in the premature death of Et2
-null mice. In a cold environment, Et2
-null mice are unable to maintain body temperature. Furthermore, hypothermia might not be the consequence of torpor, since the expression of pancreatic lipases was paradoxically decreased in the liver of Et2
-null mice (Supplemental Figure 13), whereas an increase in expression is a hallmark of torpor (44
). Nonetheless, these results deserve cautious interpretation, and this hypothesis needs to be explored further, since loss of neuronal ET-2 does not cause defects in thermoregulation.
The apparent difference in survival between constitutive and systemically inducible Et2
-null mice (Figure , C and F) may suggest the importance of ET-2 during embryonic development. However, it is highly possible that the less severe phenotype of inducible Et2-
null mice results from the gradual recombination of the floxed Et2
gene rather than the effect of ET-2 on embryonic development. Although the excision of the floxed gene can be induced relatively quickly, the efficiency still depends on time and is different in each individual (25
). Therefore, the phenotype appearance resulting from the gene’s absence will occur gradually. In our experimental setting, tamoxifen was provided to the dam after giving birth, and not during pregnancy. Therefore, the recombination efficiency of each Et2f/f
pup will show big differences depending on its milk consumption. As shown in Supplemental Figure 15, six hours after birth, intestinal ET-2 is still expressed in some neonatal Et2f/f;CAGGCre-ERTM
mice, but it is completely lost around 48 hours after birth. This result supports our hypothesis that ET-2 action in the postnatal period is essential for the growth and survival of the mice.
The results of our mouse genetic study of ET-2 provide insights into its potential implication and role in humans. In contrast to the intestine-restricted expression in rodents, human ET-2 expression has been detected in a range of tissues such as heart, lung, kidney, vasculature, ovary, and colon (46
). Currently, ET receptor antagonists such as bosentan and ambrisentan are clinically used for the treatment of pulmonary arterial hypertension. However, our data showing the possible role of ET-2 in the maintenance of lung morphology and function suggest that blocking the ET-2 signaling pathway may have adverse effects in the lungs of human patients. Therefore, the effect of ET antagonists on normal lung morphology and basal function should be examined in preclinical safety tests in clinical trials. The beneficial effect of bosentan and Ro 48-5695 in the rat trinitrobenzene sulfonic acid–induced colitis model supports the possible use of ET receptor antagonists in the treatment of human inflammatory bowel disease (IBD) (47
). However, a clear role of ET in IBD pathogenesis has not been conclusively established (50
). Unlike in rats, ET-2 is the predominant ET isoform in both mouse (I. Chang et al., unpublished observations) and human colonic mucosa (50
). Therefore, investigating a model of intestinal injury and inflammation with Et2f/f
mice would be informative to address the question of whether the beneficial effects of ET antagonists reported in the rat model are relevant to the treatment of human IBD.