Deficiency in pulmonary surfactant proteins in mice with fatty acid binding protein 4-Cre-mediated knockout of the tuberous sclerosis complex 1 gene

New findings What is the central question of this study? Does tuberous sclerosis complex 1–mammalian target of rapamycin (mTOR) signalling regulate the synthesis of surfactant proteins A and B and, if so, can this contribute to the postnatal death of Fabp4-Tsc1cKO mice? What is the main finding and its importance? Our study reveals a novel mechanism for the regulation of alveolar surfactant proteins. Tuberous sclerosis complex 1–mTOR signalling contributes to the regulation of synthesis of surfactant proteins A and B. Deficiency of tuberous sclerosis complex 1 in alveolar epithelial cells may contribute to the postnatal death of Fabp4-Tsc1cKO mice. Tuberous sclerosis complex 1 (TSC1) forms a heterodimmer with tuberous sclerosis complex 2, to inhibit signalling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). The mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as gene transcription and protein translation, in response to growth factors and nutrient signals. Originally designed to test the role of TSC1 in adipocyte function, mice in which the gene for TSC1 was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre (Fabp4-Tsc1cKO mice) died prematurely within 48 h after birth. The Fabp4-Tsc1cKO mouse revealed a much smaller phenotype relative to the wild-type littermates. Maternal administration of rapamycin, a classical mTOR inhibitor, significantly increased the survival time of Fabp4-Tsc1cKO mice for up to 23 days. Both macroscopic and microscopic haemorrhages were observed in the lungs of Fabp4-Tsc1cKO mice, while other tissues showed no significant changes. Levels of surfactant proteins A and B demonstrated a significant decrease in the Fabp4-Tsc1cKO mice, which was rescued by maternal injection of rapamycin. Co-localization of FABP4 or TSC1 with surfactant protein B was also detected in neonatal pulmonary tissues. Our study suggests that TSC1–mTORC1 may be critical for the synthesis of surfactant proteins A and B.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by development of hamartomas, which can arise in multiple organs, including brain, kidney, skin and liver (Borkowska et al. 2011). Tuberous sclerosis complex is exclusively associated with mutations in either the TSC1 or TSC2 tumour suppressor gene, which encodes the proteins hamartin and tuberin, respectively. These two proteins form a complex and function as a negative regulator for the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signalling. Interference with TSC1/2 promotes constitutive activation of mTORC1 (Hay & Sonenberg, 2004). The mTORC1 consists of mTOR, mLST8 (also termed G-protein h-subunit-like protein, GhL, a yeast homologue of LST8), raptor (regulatory-associated protein of mTOR), and PRAS40 (proline-rich Akt substrate 40 kDa), and is rapamycin sensitive (Dunlop & Tee, 2009). The mTORC1 regulates translation initiation by phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1, and functions primarily in the control of a large array of cellular processes, including cell proliferation, growth, differentiation and survival (Iadevaia et al. 2012). Recent studies also reveal that mTORC1 signalling is critical in the sensing of nutrient availability, and in the regulation of food intake and energy balance (Cota et al. 2008;Lian et al. 2008;Cota, 2009;Polak & Hall, 2009;Catania et al. 2011).
Adipose tissue, a fat storage depot and an endocrine organ, controls energy metabolism, appetite and fertility in response to nutrients and insulin. Interestingly, the fat body in Drosophila controls full-body growth in response to nutrients (Colombani et al. 2003). Downregulation of TOR signalling, most probably dTORC1, specifically in the fat body, leads to a significant reduction in overall body size (Zhang et al. 2000). A full-body knockout of any component of mTORC1 is embryonically lethal (Gangloff et al. 2004;Murakami et al. 2004). Mice with adipose-specific knockout of raptor are lean, resistant to diet-induced obesity, and demonstrate an increase in mitochondrial uncoupling in white adipose tissue (Polak et al. 2008). In addition, these mice show better metabolic parameters, including improved glucose tolerance and insulin sensitivity, as well as resistance to diet-induced hypercholesterolaemia. The higher insulin sensitivity is attributable to the leanness and to loss of the S6K1 negative feedback regulation to insulin receptor substrate 1 in adipose tissue. This phenotype is similar to that of mice lacking S6K1 , the mTORC1 positive effector, and opposite to that of mice lacking eukaryotic initiation factor 4E binding proteins 1 and 2 (Le Bacquer et al. 2007), the mTORC1 negative effectors. Consistent with these findings in the adipose-specific raptor knockout mice, rapamycin treatment causes weight loss in gerbils (Fraenkel et al. 2008) and prevents weight gain in rats and humans (Rovira et al. 2008). All these studies suggest that mTORC1 signalling in adipose tissue is critically involved in the control of whole-body energy metabolism. While most of these studies focus on the mTORC1 and its downstream effectors, little is known about the role of TSC1/2, the important upstream regulators of mTORC1, in the development and function of adipose tissues.
This study was originally designed to explore the effect of TSC1 on adipose tissues. Given that mice homozygous for Tsc1 or Tsc2 targeted mutations die by mid-embryogenesis (Kobayashi et al. 1999;Onda et al. 2002;Wilson et al. 2005), we generated a colony of mice in which the Tsc1 gene was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre, namely Fabp4-Tsc1cKO mice. Surprisingly, the Fabp4-Tsc1cKO mice died prematurely within 48 h after birth. Histological examination revealed significant haemorrhage, hyperplasia of the alveolar wall and pulmonary atelectasis. Both mRNA and protein levels of surfactant proteins (SP) A and B demonstrated a significant decrease in the Fabp4-Tsc1cKO mice. These observations suggest that deficiency in the synthesis of surfactant proteins A and B may impair the postnatal survival of Fabp4-Tsc1cKO mice.

Ethical approval
The animals used in this study were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996), and all the experimental protocols were approved by the Animal Care and Use Committee of Peking University.

Animals and animal care
Fabp4-Cre mice that express the Cre recombinase gene under the control of the Fabp4 gene promoter, as well as Tsc1 lox/lox mice, in which exons 17 and 18 of the Tsc1 gene are flanked by loxP sites by homologous recombination, were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The Fabp4-Tsc1cKO mice were generated by breeding Tsc1 lox/lox mice with Fabp4-Cre mice. Control experiments were performed using littermate Tsc1 lox/lox animals. Deletion of the Tsc1 gene was validated by the absence of its mRNA and the elevation of mTOR activity measured by the increased phosphorylation of S6 and mTOR in brown fat tissues. Mice were housed on a 12 h-12 h light-dark cycle. Normal chow and water were available ad libitum.

Rapamycin treatment
Rapamycin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was initially dissolved in 100% DMSO, stored at -20 • C, and further diluted in DMSO immediately before use. Pregnant mice were randomly divided into two groups. At the near-term pregnancy, mice were injected intramuscularly with rapamycin (1 mg (kg body weight) −1 day −1 ) or vehicle for 3 days before the birth of pups. Administration of rapamycin to mother mice at the same dose continued for 2 days after labour. Neonatal mice were closely observed for life signs to assess the survival of the offspring.

Extraction of RNA and quantitative real-time PCR analysis
Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription and quantitative real-time PCR were performed as previously described (Li et al. 2008;Xu et al. 2009). The PCRs were performed in duplicate, and each experiment was repeated between three and five times. Primers used in this study are shown in Table 1.

Immunostaining
The lungs were quickly removed and prepared for immunostaining as previously described (Xu et al. 2009). Slides were individually incubated with TSC1 (1:100 dilution; CST) or phosphorylated mTOR (p-mTOR; Ser 2448) antibody (1:100 dilution; CST) in a humid chamber at 4 • C overnight. Secondary antibody staining was performed with a biotin-labelled horse anti-rabbit antibody (1:200 dilution) for 1 h at room temperature, followed by incubation with a streptavidin-biotin horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was detected using diaminobenzidine substrate (peroxide substrate kit, SK-4100; Vector Laboratories) for 2-5 min. Slides were then counterstained with Mayer's Haematoxylin before dehydration and mounting. The expressions of TSC1 and p-mTOR were analysed by comparing the staining intensities between different samples with and without the addition of a primary antibody in the same conditions.

Double immunostaining
Paraffin-embedded sections were incubated overnight with a mixture of rabbit polyclonal antibodies to FABP4 (1:100 dilution) or TSC1 (1:100 dilution) and mouse monoclonal antibody to SP B (1:50 dilution), then incubated with the mixture of the following secondary antibodies: goat anti-rabbit fluorescein isothiocyanate-conjugated IgG (1:100 dilution) and goat anti-mouse Texas Red-conjugated IgG (1:100 dilution). Controls included substitution of primary antibodies with mouse IgG or rabbit IgG. The FABP4 or TSC1 and SP B positive cells were evaluated using Image Plus software (fCoder Group, Inc., Alexandria, VA, USA) to quantify the fluorescence intensity inside the cells. Cells with fluorescence intensity ≥150% of background were considered positive. A total of 15 tissue sections from five mice were evaluated.

Statistical analysis
Data are expressed as means ± SEM. Data analysis used GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). One-way ANOVA, Student-Newman-Keuls test (comparisons between multiple groups) or Student's unpaired t test (between two groups) was used as appropriate. A value of P < 0.05 denotes statistical significance.

Postnatal death of Fabp4-Tsc1cKO mice and the rescuing effect of rapamycin
As shown in Fig. 1, Fabp4-Tsc1cKO mice were significantly smaller relative to their littermates ( Fig. 1A and B), while the body length showed no difference (Fig. 1C). Postnatal death occurred in all Fabp4-Tsc1cKO mice. All mice died prematurely within 48 h after birth, with over 60% of death occurring during the first 24 h (Fig. 1D). In order to determine the specific effect of Tsc1 gene deletion in tissues expressing FABP4, we examined the rescuing effect of rapamycin, a specific inhibitor of mTOR signalling, on the survival of Fabp4-Tsc1cKO mice. As shown in Fig. 1E, administration of rapamycin at a dose of 1 mg kg −1 day −1 to the near-term pregnant mice significantly increased the survival rate of Fabp4-Tsc1cKO mice. The maximal survival time was extended up to 23 days (Fig. 1E).

Expression of FABP4 in neonatal mice and adult mice
A series of studies were next performed to determine the potential cause of postnatal death in Fabp4-Tsc1cKO mice. We first examined the tissue expression of FABP4. Differential expression of FABP4 was observed in neonatal and adult mice. In adult mice, FABP4 expression was limited to being present only in brown and white adipose tissues ( Fig. 2A). In contrast, neonatal mice demonstrated a distribution of FABP4 in a wide range of tissues (Fig. 2B).
In addition to brown and white adipose tissues, FABP4 was detected in lung, heart, skin and kidney, and in liver and brain at a lower level in neonatal mice.

Pulmonary lesions in Fabp4-Tsc1cKO mice
We next examined the histological changes in FABP4expressing tissues derived from the neonatal FABP4-Tsc1cKO mice. No significant histological alteration was observed in tissues such as heart (Fig. 3A), liver (Fig. 3B), skin (Fig. 3C), kidney (Fig. 3D) and pancreas (Fig. 3E). However, macroscopic haemorrhage was observed in the lungs (Fig. 4A). Haematoxylin and Eosin staining confirmed significant pathological lesions in pulmonary tissues, including haemorrhage, hyperplasia of the alveolar walls and pulmonary atelectasis (Fig. 4B). Given that hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) have been reported to be associated with haemorrhage, we next examined the expression of HIF-1α and VEGF in the lungs of Fabp4-Tsc1cKO mice. As shown in Fig. 4C and D, mRNA levels of HIF-1α and VEGF were markedly increased in the pulmonary tissues in Fabp4-Tsc1cKO mice relative to the wild-type animals.

Expression of TSC1 and p-mTOR in the lung of Fabp4-Tsc1cKO mice
In order to validate the deletion of Tsc1 in the pulmonary tissue of Fabp4-Tsc1cKO mice, we next examined the expression of TSC1 and p-mTOR by immunostaining. As shown in Fig. 5A, TSC1 immnoreactivity was detected in the pulmonary epithelial cells, whereas no positive signal was demonstrated in the Fabp4-Tsc1cKO mice. Consistent with the change in the TSC1 level, Fabp4-Tsc1cKO mice demonstrated a much stronger signal for p-mTOR staining in the alveolar epithelial cells (Fig. 5B). These results suggest that TSC1 inactivation enhances mTOR activity in the pulmonary epithelia.

Fabp4-Tsc1cKO mice
Given that surfactant proteins secreted by type II alveolar epithelial cells play a central role in pulmonary ventilation, levels of alveolar SPs were then examined. As shown in Fig. 6A, mRNA levels of pulmonary SP A and SP B were markedly decreased in the pulmonary tissues derived from the neonatal Fabp4-Tsc1cKO mice, whereas SP C demonstrated no significant change. This change was associated with a significant reduction in the protein levels of both SP A and SP B (Fig. 6B).
Administration of rapamycin at a dose of 1 mg kg −1 day −1 into the near-term pregnant mice prevented the reduction of SP A and SP B mRNA (Fig. 6C). To further test the effect of mTOR signalling in the regulation of SP A and SP B transcription, A549 cells, a human alveolar basal epithelial cell line, were transfected with mTOR-WT or mTOR-KD plasmids to increase and decrease the mTOR signalling in these cells, respectively. As a control, GFP-pcDNA3.0 was used. As shown in Fig. 6D, mRNA levels of SP A and SP B were markedly decreased after activation of mTOR by mTOR-WT plasmid, whereas inhibition of mTOR by mTOR-KD plasmid increased the mRNA levels of SP A and SP B.

Co-localization of FABP4 or TSC1 with surfactant protein B in alveolar epithelium
Double immunofluorescent staining was performed to colocalize the expression of FABP4 or TSC1 with SP B in the neonatal lung tissues. As shown in Fig. 7A, antibody recognizing FABP4 showed a positive reaction in the alveolar epithelial cells that were also reactive for SP B. Likewise, co-localization of TSC1 immunoreactivity with SP B was observed in the alveolar epithelial cells (Fig. 7B). Control antibodies produced no positive signal.

Discussion
Our present study demonstrates that conditional deletion of Tsc1 driven by the Fabp4 promoter causes a deficiency in the synthesis of surfactant proteins A and B, which may at least partly contribute to the postnatal death of Fabp4-Tsc1cKO mice. This conclusion is supported by the following observations: (i) Fabp4-Tsc1cKO mice die within 48 h after birth; (ii) inhibition of mTORC1 signalling by rapamycin rescues the mice from postnatal death; (iii) a much wider expression of FABP4 is found in neonatal tissues, including the lungs; (iv) levels of alveolar surfactant proteins A and B are significantly decreased in Fabp4-Tsc1cKO mice; (v) there exists a negative relationship between mTOR signalling and SP A and SP B transcription in the cultured alveolar epithelial cells; and (vi) FABP4 or TSC1 co-localizes with surfactant protein B in alveolar epithelial cells of the neonatal lungs.
Fatty acid binding protein 4, also known as adipocyte-FABP, was first detected in mature adipocytes and adipose tissue (Reese-Wagoner et al. 1999). This protein was originally termed adipocyte P2 (aP2) because of its high sequence similarity (67%) to peripheral myelin protein 2 (M-FABP/FABP8). Originally identified as an adipocytespecific protein (Spiegelman & Green, 1980), the promoter of Fabp4 has been widely used to target adipose tissuespecific gene expression in mice (Ross et al. 1990;Barlow et al. 1997;He et al. 2003). Two different lines of Fabp4-Cre mice have been used in most published studies. One was developed in Dr Barbara Kahn's laboratory at Harvard University (Abel et al. 2001) and the other in Dr Ronald Evans's laboratory at the Salk Institute (He et al. 2003). In both lines, Cre activity is expressed in white and brown adipose tissues in adult mice. Our study used the latter line, which is commercially available from Jackson Laboratory (stock no. 005069).
Most Fabp4-Cre-mediated gene knockout mice can survive, but there also are some exceptions. Ablation of the Vhl gene in adipose driven by the Fabp4 gene promoter exhibits embryonic lethality at embyonic day 14.5-18.5, which results from haemorrhages in the brain and liver induced by the upregulation of HIF-1α/VEGF activity (Zhang et al. 2012). In addition, Mudhasani et al. (2011) showed that mice with Fabp4-Cre-driven deletion of dicer gene typically died within 3 weeks after birth, shortly before weaning. These results suggest an expression of FABP4 outside the adipose tissues. Indeed, recent studies have demonstrated a wider expression of FABP4 in an array of cells and tissues, including macrophages upon their differentiation from monocytes, human bronchial epithelial cells, arterial endothelial cells, trophoblasts, liver, skeletal muscle fibres, lipoblastoma and liposarcoma, and human urothelial carcinomas (Shum et al. 2006;Biron-Shental et al. 2007;Lee et al. 2007;Ferrell et al. 2008;Boiteux et al. 2009;Elmasri et al. 2009;Scifres et al. 2011). Consistent with these findings, our study suggests that FABP4 is present in a wide range of tissues during development and becomes adipocyte specific in adulthood. Taken together, all these findings may challenge the previous concept on the use of Fabp4 promoter to target adipose tissue-specific gene expression during development.

Figure 4. Pulmonary lesions of the Fabp4-Tsc1cKO mice
The lungs were taken from neonatal Fabp4-Tsc1cKO mice (KO) and wild-type littermates (WT). A, photographs were taken under the stereoscope. Macroscopic haemorrhage was observed in the lungs of the Fabp4-Tsc1cKO mouse. B, Haematoxylin and Eosin staining of lung tissue sections shows pulmonary lesions, including haemorrhage, hyperplasia of the alveolar walls and pulmonary atelectasis. The mRNA levels of hypoxia-inducible factor-1α (HIF-1α; C) and vascular endothelial growth factor (VEGF; D) were markedly increased in the pulmonary tissues in Fabp4-Tsc1cKO mice relative to the wild-type animals. * P < 0.05, * * P < 0.01 (n = 6). In our animal model of Fabp4-Tsc1cKO mice, specific deletion of the Tsc1 gene driven by Fabp4 promoter causes postnatal death. Conditional deletion of Tsc1 driven by the Fabp4 promoter is confirmed by the absence of TSC1 mRNA and activation of mTOR as measured by increased phosphorylation of S6 and mTOR in the brown adipose tissues (data not shown). Previous studies have demonstrated that either TSC1 or TSC2 is critical for

Figure 5. Expression of tuberous sclerosis complex 1 (TSC1) and phosphorylated mammalian target of rapamycin (p-mTOR)in the lungs of Fabp4-Tsc1cKO mice
The immunoreactivity for TSC1 (A) and p-mTOR (B) is shown in the lung tissues. A specific signal was observed in the alveolar epithelial cells. Relative to the wild-type mice, TSC1 immunoreactivity was significantly reduced, whereas the p-mTOR signal intensity was higher in the pulmonary tissues of Fabp4-Tsc1cKO mice. survival (Tomasoni & Mondino, 2011). Mice homozygous for Tsc1 targeted mutations die by mid-embryogenesis, while a significant number of heterozygous (Tsc1 +/− ) mice on the C57BL/6 background die in the postnatal period, normally at 1-2 days, from unknown causes (Wilson et al. 2005). In humans, mutation of either the Tsc1 or the Tsc2 gene causes tuberous sclerosis complex, a multisystem genetic disease, which is commonly characterized by neurological symptoms, such as seizures, autism, mental retardation and learning disabilities. While neuronal dysfunction is common in tuberous sclerosis complex, it is unlikely that the dysfunction of the central nervous system is the leading cause of embryonic or neonatal death. In a mouse tuberous sclerosis epilepsy model, conditional deletion of Tsc1 in astrocytes induces epilepsy commonly observed in tuberous sclerosis complex patients, and these mice survive until adulthood (Uhlmann et al. 2002). The present study reveals a novel mechanism for the postnatal death of Fabp4-Tsc1cKO mice. Significant pathological changes, which include haemorrhage, hyperplasia of the alveolar walls and pulmonary atelectasis, are observed in these mice. The pulmonary haemorrhage may be due to the defect in the blood vessel structure induced by HIF-1α activation. A high level of HIF-1α has been reported to stimulate the expression of VEGF, which subsequently increases capillary permeability (Weis & Cheresh, 2005;Zhang et al. 2012). Neonatal respiratory distress syndrome is caused by inadequate amounts of pulmonary surfactant due to delayed lung development (Avery & Mead, 1959). Surfactant, a lipoprotein comprised of phospholipids (∼80%), cholesterol (∼10%) and proteins (∼10%), functions to reduce surface tension and to prevent alveolar collapse at end expiration (Weaver & Whitsett, 1991). Surfactant deficiency or dysfunction is associated with the occurrence and development of many pulmonary diseases, such as neonatal respiratory distress syndrome, acute respiratory distress syndrome, asthma and chronic obstructive pulmonary disease (Wright, 2003). Four types of surfactant proteins (A, B, C and D) have been reported (Weaver & Whitsett, 1991). Surfactant protein A is the most abundant, while SP B is crucial for the adsorption of the surfactant film at the alveolar air-liquid interface (Whitsett & Weaver, 2002). Surfactant protein B has been demonstrated to be critical for normal gas exchange in the perinatal period and is therefore indispensable for neonatal survival.
Our study suggests that the neonatal death of Fabp4-Tsc1cKO mice may result from a deficiency in the synthesis of SP A and SP B in Fabp4-Tsc1cKO mice. First, both the mRNA and the protein levels of SP B are markedly reduced in the Fabp4-Tsc1cKO mice. Maternal administration of rapamycin prevents the reduction of SP B induced by the activation of mTOR signalling and significantly extends the survival time of the Fabp4-Tsc1cKO mice. Second, an alteration in mTOR signalling directly modulates the transcription of SP B in cultured alveolar epithelial cells. Third, FABP4 or TSC1 co-localizes with SP B in the alveolar epithelial cells. It is well known that type II cells synthesize SP A and SP B in the lung. Our studies indicate that TSC1-mTOR signalling may simultaneously modulate the synthesis of both SP A and SP B in these cells. Taken together, all these observations suggest that TSC1 may be involved in the regulation of SP A and SP B, and specific deletion of Tsc1 driven by the Fabp4 promoter may impair the synthesis of SP A and SP B in the alveolar epithelial cells. This finding is consistent with the previous reports by Ikeda et al. (2011), in which aberrant activation of the Akt-mTOR signalling pathway in lung epithelium causes infant respiratory distress syndrome, and by Miakotina et al. (2002), in which the rapamycin-sensitive phosphoinositide 3-kinase-S6K1 signalling pathway is demonstrated to mediate insulininduced inhibition of SP A mRNA levels in lung epithelial cells.
In conclusion, our studies suggest a novel mechanism for the regulation of alveolar surfactant proteins. Tuberous sclerosis complex 1 contributes to the regulation of synthesis of surfactant proteins A and B. Deficiency of TSC1 in alveolar epithelial cells may partly contribute to the postnatal death of Fabp4-Tsc1cKO mice.