Abstract
ErbB4 receptor and thyroid transcription factor (TTF)-1 are important modulators of fetal alveolar type II (ATII) cell development and injury. ErbB4 is an upstream regulator of TTF-1, promoting its expression in MLE-12 cells, an ATII cell line. Both proteins are known to promote surfactant protein-B gene (SftpB) and protein (SP-B) expression, but their feedback interactions on each other are not known. We hypothesized that TTF-1 expression has a feedback effect on ErbB4 expression in an in-vitro model of isolated mouse ATII cells. We tested this hypothesis by analyzing the effects of overexpressing HER4 and Nkx2.1, the genes of ErbB4 and TTF-1 on TTF-1 and ErbB4 protein expression, respectively, as well as SP-B protein expression in primary fetal mouse lung ATII cells. Transient ErbB4 protein overexpression upregulated TTF-1 protein expression in primary fetal ATII cells, similarly to results previously shown in MLE-12 cells. Transient TTF-1 protein overexpression down regulated ErbB4 protein expression in both cell types. TTF-1 protein was upregulated in primary transgenic ErbB4-depleted adult ATII cells, however SP-B protein expression in these adult transgenic ATII cells was not affected by the absence of ErbB4. The observation that TTF-1 is upregulated in fetal ATII cells by ErbB4 overexpression and also in ErbB4-deleted adult ATII cells suggests additional factors interact with ErbB4 to regulate TTF-1 levels. We conclude that the interdependency of TTF-1 and ErbB4 is important for surfactant protein levels. The interactive regulation of ErbB4 and TTF-1 needs further elucidation.
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Background
Surfactant deficiency causes neonatal respiratory distress syndrome (RDS) and the development of bronchopulmonary dysplasia (BPD), both severe diseases of immature lungs in preterm born infants (Northway 1967). While the severity of RDS in preterm born infants has decreased in recent decades with better pre- and postnatal care including the application of antenatal corticosteroids, postnatal surfactant supplementation, and more gentle modes of mechanical ventilations (Jobe 1993), the incidence of BPD has only minimally changed (Kair 2012). Despite the growing knowledge of inflammatory (Bose 2008) and oxidative injury (Saugstad 2003) contributing to BPD pathogenesis, treatment options for BPD still have limited success (Beam 2014). Increasing knowledge of the regulation of growth and transcription factor signaling impacting surfactant protein levels in RDS and lung remodeling in BPD should contribute important insights into new therapeutic strategies to prevent or treat BPD.
ErbB receptors are important regulators of cell proliferation, maturation and differentiation (Gassmann 1995). In the fetal lung, ErbB4 protein is highly expressed in fetal alveolar type II (ATII) cells (Liu 2007) and is necessary for the timely initiation of surfactant expression and synthesis (Dammann 2003) and the progression of morphologic lung development (Liu 2010). ErbB4 is unique in the ErbB family of tyrosine kinase receptors, since it can be cleaved by presenillin (Hoeing 2011; Fiaturi 2014), expresses strong nuclear signaling (Williams 2004) and interacts with transcription factors such as Stat5A (Zscheppang 2011) and TTF-1 (Zscheppang 2013) that are important in promoting Sftpb expression in ATII cells.
TTF-1 is a nuclear transcription factor that activates transcription of genes in lung, brain and thyroid (Bingle 1997). TTF-1 plays an important role in the developing lung by regulating branching morphogenesis (Minoo 1995) and surfactant protein gene expression (DeFelice 2003). The localization of Nkx2.1 – the gene encoding for TTF-1 – expression in human fetal lung tissue follows the pattern of distribution of Sftpb, and is down regulated in pathologic situations like inflammation (Stahlman 1996). Surfactant Protein B (SP-B), the protein product of the SftpB gene, is the most critical functional component of surfactant for lowering surface tension (Perez-Gil 2008). Heterozygous dysfunction of Nkx2.1 leads to respiratory dysfunction and recurrent pulmonary infections likely due to decreased SP-B levels (Devriendt 1998). The regulation of Nkx2.1 expression is only partially understood (Hamdan 1998).
Little is known about the regulation of ErbB4 and TTF-1 signaling mechanisms in the fetal lung around the time of initiation of surfactant production or about their interactions with each other. We hypothesized that expression of ErbB4 and TTF-1 proteins are regulated in a feedback loop to coordinate their mutual activity on regulating Sftpb expression. We here show negative feedback regulation between TTF-1 and ErbB4, and speculate that TTF-1 plays a key role in compensating for ErbB4 loss to maintain Sftpb expression.
Materials and methods
Materials
The immortalized mouse lung alveolar epithelial cell line MLE-12 was obtained from the American Type Culture Collection (Manassas, VA); time-dated pregnant wild type Swiss Webster Mice were obtained from Taconic (Hudson, NY). Rabbit polyclonal TTF-1 antibody (H-190) was obtained from Abcam (Cambridge, MA), rabbit polyclonal ErbB4 antibody (C-18) and rabbit polyclonal SP-B antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal β-actin antibody, IRDye 680LT goat anti-rabbit IgG antibody (H + L) and IRDye 800CW and goat anti-mouse antibody IgG (H + L) were obtained from Li-Cor (Lincoln, NE). Bovine serum albumin (BSA) was obtained from Sigma (St.Louis, MO); Dulbecco’s Modified Eagles Medium (DMEM) and Ham’s F12 culture media were obtained from Invitrogen (Grand Island, NY); fetal bovine serum (FBS) was from Thermo Fisher Scientific (Waltham, MA); Plasmid Midi Kit was from Qiagen (Germantown, MD). FuGene® HD Transfection Reagent was obtained from Promega (Madison, WI); Dispase was from BD Bioscience (Franklin Lakes, NJ); Collagenase type 2 was obtained from Worthington (Lakewood, NJ), Trypsin 1:250 was from USB Corporation (Cleveland, OH) and Desoxyribonuclease I was from Sigma Aldrich (St. Louis, MO); Ketamine hydrochloride was from Fort Dodge Animal Health (Fort Dodge, IA) and Rompun® (Xylazine) was obtained from Bayer Agriculture Division (Shawnee Mission, KS). DAPI was from Vector Laboratories (Burlingame, CA). pEGFP N3 (control), and pHER4 (full length human ErbB4 receptor) plasmids (Lee 2002; Williams 2004) were used as previously published (Zscheppang 2011). pRC/CMV/Nkx2.1 (Nkx2.1 expression plasmid) was kindly provided by Dr. Jeffrey Whitsett (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH) (Zhou 2008).
Preparation of primary fetal mouse ATII epithelial cell cultures
All animal use was performed according to an animal research protocol approved by the institutional IACUC. Primary ATII cells were freshly isolated from time-dated pregnant Swiss Webster mice as previously described (Zscheppang 2013). Briefly, pregnant Swiss Webster mice were sacrificed at E17.5 of gestation by CO2 inhalation followed by cervical dislocation. Fetal lungs were removed from the isolated fetuses, washed in sterile HBSS, minced with a razorblade, and incubated with collagenase type II diluted in serum-free DMEM for 2 h at 37 °C. The reaction was stopped on ice for 30 min. Cells were centrifuged and resuspended in DMEM. After a second centrifugation the pellet was resuspended in DNase and trypsin and incubated for 12 min at 37 °C. The reaction was stopped by DMEM containing 10 % fetal calf serum (FBS). The cells were filtered through a 40 μm nylon filter, centrifuged, resuspended in DMEM containing 10 % FBS, and plated in culture flasks for 60 min at 37 °C (21 %02/5 %C02) to allow for differential adherence of lung fibroblasts. For ATII cell isolation the supernatant from the first differential adherence was centrifuged, the cell pellet resuspended in DMEM containing 10 % FBS, and plated again in the same conditions for a second differential adherence. Supernatants were removed and centrifuged. The cell pellet was resuspended and cells were plated in 6-well plates in DMEM containing 20 % FBS. After 24 h of incubation 200 μg of cis-4-Hydroxy-L-Proline was added to each well for another 24 h to minimize the proliferation of the residual fibroblasts (Kao 1977). Wells were washed three times with PBS before transfection experiments were started.
Preparation of primary ATII epithelial cell cultures from adult male ErbB4-transgenic mice
HER4heart(−/−) mice (hereafter designated HER4heart mice), a transgenic mouse line in which fetuses homozygous for ErbB4 deletion were rescued from their lethal cardiac defects by expressing a human ErbB4 (HER4) cDNA under the cardiac-specific α-myosin heavy chain promoter (MHC) (Tidcombe 2003), were used for these experiments. Mice were genotyped by PCR (Supplemental Fig. 1A), and the absence of ErbB4 receptors was confirmed in lung fibroblasts (Supplemental Fig. 1B). All animals were housed in a pathogen-free animal facility at Tufts University. Primary ATII cells were freshly isolated from adult male HER4heart mice as previously published with slight changes (Corti 1996; Warshamana 2001). Briefly, mice were narcotized with a ketamine-xylazine solution (153 mM ketamine, 24.5 mM xylazine). After rinsing the lung blood vessels free from cells with PBS, 1 mL of dispase was installed into the lung via the trachea. The lung was removed from the mouse and incubated in dispase on ice for 40 min, minced with a razorblade, incubated with DNase in DMEM at 37 °C for 10 min, sequentially filtered through 100, 40, and finally 20 μm nylon filters, centrifuged and resuspended in DMEM containing 10 % FBS. For differential adherence of fibroblasts, cells were plated in culture plates for 60 min at 37 °C (21 %02/5 %C02). For ATII cell isolation, the supernatant was collected, centrifuged, and the resuspended pellet plated for a second and third differential adherence under the same conditions for 45 and 30 min, respectively. After the third differential adherence, cells were centrifuged, the pellet was resuspended in DMEM containing 10 % FBS, and cells were plated in one well of a 6-well plate. After 48 h of incubation, 200 μg of cis-4-Hydroxy-L-Proline was added to each well for another 24 h to inhibit the proliferation of the residual fibroblasts (Kao 1977). Cells were grown on plastic in DMEM for another 24 h before harvesting. Growth conditions for fetal and adult ATII cells were similar, since they conserve the expression of ATII cell markers in both cell types (Dey Hazra 2011).
Transfection experiments
To examine the effects of HER4 and Nkx2.1 (the genes encoding for ErbB4 and TTF-1) overexpression on SP-B, ErbB4, and TTF-1 protein expression levels, MLE-12 cells and primary fetal ATII cells were transfected with plasmid expressing human ErbB4 (HER4), or Nkx2.1, or an empty control construct (EGFP) (Zhou 2008; Zscheppang 2011, 2013). The MLE-12 cells were only transfected with Nkx2.1 or EGFP, since we have already published the effects of ErbB4 overexpression in MLE-12 cells on surfactant protein expression (Zscheppang 2013). The cells were transfected for 24 h using FuGene® transfection reagent in accordance with the manufacturer’s suggestions. Briefly, cells were grown on 6-well plates up to 30–50 % confluence for primary ATII cells and MLE-12 cells, respectively. Two micrograms of DNA were diluted in serum free medium and FuGene® reagent and incubated for 15 min at room temperature before the mixture was added drop-wise into the serum-containing media. Primary ATII cells were grown in DMEM containing 20 % FBS. MLE-12 cells were grown in serum-supplemented (2 %) HITES medium. To quantify the transfection efficiency, the numbers of green fluorescent EGFP positive cells and total DAPI stained cells were counted under fluorescence microscopy. The transfection efficiency was 30–40 % in MLE 12 cells (Supplemental Fig. 2A), and about 20 % in primary fetal ATII cells (Supplemental Figure 2B).
Western blotting
After 24 h of incubation the cells were washed three times with PBS (pH 7.2; 137 mM NaCl, 10.14 mM Na2HPO4, 2.7 mM KCl, 1.76 mM KH2PO4) and scraped in 100–300 μL lysis buffer per well (20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 % NP-40, 1 mM Na3VO4, 4.5 mM Na4P2O7, 10 mM β-glycerol phosphate, 1 mM Na3VO4, 1 mM ZnCl2, 1 mM NaF, 1 mM PMSF and 10 μg each of aprotinin, leupeptin and pepstatin). After 15 min of incubation on ice, cells were scraped and lysates were cleared by microcentrifugation for 15 min at 4 °C. Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA) was used to determine the concentration of total protein. To detect individual proteins, cell lysates with an amount of 30 ng of total protein were boiled in Laemmli buffer for 5 min at 100 °C. Proteins were separated by 7 % SDS polyacrylamide gel electrophoresis and transferred to a 0.45 μm nitrocellulose membrane. For SP-B and TTF-1 detection, a 9 % SDS polyacrylamide gel and a 0.2 μm membrane were used. Blots were blocked in 1 % BSA and incubated with antibodies against individual proteins. Proteins were visualized by scanning the membranes with a Li-Cor® Odyssey infrared imaging system. Membranes were stripped in stripping buffer (pH 2; 25 mM glycine and 52 mM SDS) for 30 min at 50 °C and reprobed up to a maximum of 5 times.
Data analysis
All protein expressions of ErbB4, TTF-1, or SP-B were measured by densitometry, normalized to the control condition (set to 100 %) and presented as mean ± SEM. The results of all transfection experiments were also expressed as percentage of their EGFP-transfected experiment-specific control (set to 100 %). The effect of ErbB4 depletion in adult homozygous mutant HER4heart mice on SP-B and TTF-1 protein expression was expressed as percentage of the expression of the heterozygous controls (set to 100 %). Statistical significance was evaluated using a paired two-tailed t-test except for one instance when the a priori hypothesis tested was a one-way hypothesis.
Results
Nkx2.1 overexpression increases SP-B, but decreases ErbB4 protein expression in MLE-12 cells
To determine the effect of Nkx2.1 overexpression in MLE-12 cells only experiments which showed an adequate (>400 %) increase of TTF-1 protein expression were analyzed. These experiments showed increased TTF-1 protein expression to 647 ± 76 % of controls (N = 8, P = 0.0002) (Fig. 1a). This was associated with a significant increase of SP-B protein expression to 130 ± 10 % (N = 8, P = 0.022) and a significant decrease of ErbB4 protein expression to 72 ± 4 % (N = 8, P = 0.0003) (Fig. 1b) when compared to the specific EGFP-transfected control cells.
Nkx2.1 overexpression increases SP-B, but decreases ErbB4 protein expression in primary ATII cells
In fetal mouse ATII cells only experiments which showed an adequate (>150 %) increase of TTF-1 protein expression were analyzed. In these experiments Nkx2.1 overexpression increased TTF-1 protein expression to 224 ± 43 % (N = 8, P = 0.023) (Fig. 2a), resulting in a similar significant increase in SP-B protein expression to 151 ± 18 % (N = 7, P = 0.03) and a significant decrease in ErbB4 protein expression to 75 ± 3 % (N = 8, P = 0.0001) (Fig. 2b) when compared to EGFP-transfected control cells.
HER4 overexpression increases SP-B and TTF-1 protein expression in fetal mouse ATII cells
Only experiments which showed an adequate (>150 %) increase of ErbB4 protein expression were analyzed. HER4 overexpression increased ErbB4 protein expression in fetal mouse ATII cells to 186 ± 13 % (N = 6, P = 0.0013) (Fig. 3a). This was associated with a significant increase in SP-B protein expression to 133 ± 1.7 % (N = 6, P = 0.0001) and in TTF-1 protein expression to 178 ± 28 % (N = 5, P = 0.05) (Fig. 3b) when compared to EGFP-transfected control cells.
ErbB4 deletion increases TTF-1 protein expression but does not affect SP-B protein expression in adult male HER4heart ATII cells
In primary male adult HER4heart ATII cells, genetic deletion of ErbB4 was associated with a significant increase in TTF-1 protein expression to 174 ± 14 % (N = 14, P = 0.0002) when compared to heterozygote control cells (100 ± 9 %, N = 14, 100 ± 11 %, N = 8, respectively). However, the loss of ErbB4 did not affect SP-B protein expression (103 ± 15 %, N = 8, P = 0.86) (Fig. 4).
Discussion
The TTF-1 transcription factor is a crucial regulator of the development of the lung, the brain, and the thyroid (Bingle 1997). In the lung, acute inflammation, edema, hemorrhage, and atelectasis inhibit expression of Nkx2.1, the gene encoding for TTF-1, while in regeneration processes (Stahlman 1996) or in compensatory growth (Takahashi 2010), TTF-1 protein levels are increased, indicating the importance of TTF-1 in lung growth and remodeling. In acute inflammation, cytokines like TNF-α have been shown to decrease TTF-1 proteins at the transcriptional level (Das 2011), but little is known about how Nkx2.1 expression is otherwise regulated and if expression feedback mechanisms are present. We have previously shown that TTF-1 functions as a downstream signal for ErbB4-mediated upregulation of Sftpb expression (Zscheppang 2013). Here we show that TTF-1 provides feedback regulation on ErbB4 protein expression. Our results also suggest that elevated TTF-1 levels may maintain SftpB expression to compensate for longstanding ErbB4 deletion.
TTF-1 is a tumor marker for thyroid and lung carcinomas (Moldvay 2004). The role of TTF-1 in lung cancer cells is not fully understood, but TTF-1 is an apparent positive prognostic marker in adenocarcinomas of the lung (Berghmans 2006). In non-small cell lung carcinoma cells, Nkx2.1 overexpression represses the expression of the cell cycle protein Ki-67 and may serve as a tumor suppressor gene (Zu 2012). It also represses Muc5b, one of the major mucin genes of the respiratory tract, which is highly expressed in human lung adenocarcinoma (Jonckheere 2010). On the other hand, Nkx2.1 promotes survival of cancer cells if the gene is amplified, functioning as a lung cancer specific oncogene (Kwei 2008), while Nkx2.1 suppression inhibits cell proliferation of lung adenocarcinoma cells leading to apoptosis (Tanaka 2007). This “zwitter function” of TTF-1 is paralleled by similar zwitter functions of ErbB4 in cancer biology, in which ErbB4 expression is associated with either tumor progression or with good prognosis (Gullick 2003). The functions of ErbB4 and of TTF-1 in lung development initiated our focus on their interactions in developing alveolar ATII cells.
In our initial approach, we used the MLE-12 cell line, a mouse adult lung alveolar epithelial cell line that has been frequently used to study the regulation of surfactant protein production (Wikenheiser 1993). We found that the transfection efficiency of MLE-12 cells is twice that of primary fetal ATII cells. However, our data here indicate that despite the lower efficiency of transfection observed in primary fetal ATII cells the effects of Nkx2.1 and HER4 overexpression on SP-B protein expression are similar in both cell types. This difference in transfection efficiency is consistent with other reports (Marsh 2009) and is likely due to a higher replication rate seen in cell lines like MLE-12 cells. When comparing the phenotypical behavior of primary adult with fetal ATII cells it seemed to be more difficult to grow primary adult ATII cells, resulting in an unpredictable and widely variable viability in these different kinds of primary cells (Hamm 2002). We have used fetal primary cells for our transfection experiments, despite the fact that adult primary cells may be closer in their character to the MLE-12 cells, since MLE-12 cells were derived from adult lungs. In addition, fetal cells grow more efficiently and are therefore easier to transfect than their adult primary cell counterparts. To gain cell culture material from adult animals needs to sacrifice a lot of animals and we tried to keep the number of sacrificed animals low.
The mature SP-B protein has a size of 8 kDa. For our experiments we detected the SP-B proprotein (pro SP-B) with a size of 40 kDa due to clearer signals. The pro SP-B can show split (Figs. 1b, 3b and 4) or single (Fig. 2b) signal bands. Depending on the maturational status of the cell cycle the proprotein can show processing intermediates (Brasch et al. 2004). Inconsistent western blot bands can also be caused by spicing variants of SP-B (Chi et al. 1998). This phenomenon is known for TTF-1 as well (Li 2000) even though our experiments showed only single band signals for TTF-1.
ErbB4 is known to be important for differentiation of ATII cells of the developing lung (Dammann 2003), the mammary gland, the nervous system, and the heart (Carpenter 2002). In the lung, both TTF-1 and ErbB4 protein have important roles in promoting surfactant protein expression (Boggaram 2009; Zscheppang 2011). Our previous experiments in fetal murine ATII cells showed that there is an interaction between TTF-1 and ErbB4 (Zscheppang 2013). We here show evidence of a negative feedback loop between ErbB4 and TTF-1, in that overexpression of Nkx2.1 decreased ErbB4 protein expression. Negative feedback loops are well-documented for several hormones, for example thyroid-stimulating hormone and thyroxine (Shupnik 1989) or the adrenocorticotropic hormone and cortisol (Keller-Wood 1984). However, the feedback relationship of ErbB4 on TTF-1 appears more complex. TTF-1 protein levels were increased by ErbB4 overexpression in fetal ATII cells and MLE-12 cells, consistent with the proposed negative feedback relationship. However, the opposite was seen in HER4heart ATII cells, which showed evidence of positive feedback, i.e., the lack of ErbB4 was associated with increased TTF-1 protein levels. This suggests that the feedback effects of ErbB4 on TTF-1 protein are more complex and likely involve additional components to mediate the full effect. Nevertheless, our data clearly show that ErbB4 levels do affect the levels of TTF-1 protein. More studies are needed, including gene expression studies, in order to further characterize the mechanisms involved in the mutual feedback relationships of TTF-1 and ErbB4 in their roles of orchestrating surfactant protein production.
We used the HER4heart ATII cells in an attempt to gain further insight into the importance of the regulatory feedback between TTF-1 and ErbB4. We have previously shown that pulmonary ErbB4 deletion leads to a lung phenotype in fetal and adult animals, including a delayed onset of fetal surfactant synthesis and Sftpb expression (Liu 2010), accompanied by decreased alveolar space and increased thickness of alveolar septae, mirroring the pathologic picture of bronchopulmonary dysplasia. However, these lungs exhibit catch-up expression on the effects of ErbB4 deletion at term, resulting in normal Sftpb gene expression in adult animals (Purevdorj 2008). While Sftpb gene expression is normalized in ErbB4-deleted adult lungs, other components of lung development remain abnormal, including evidence of alveolar simplification combined with evidence of chronic inflammation, a hyperreactive airway system and surfactant protein D downregulation (Purevdorj 2008). Thus, while our observed relationship between ErbB4 loss and TTF-1 expression was unexpected and differed from that in fetal ATII cells, these results provide more evidence of the importance of normal ErbB4 expression in lung ATII cells for normal cellular function.
We here studied the effect of TTF1-ErbB4 interactions on SP-B protein levels. This is just one element of surfactant homeostasis, and so these studies do not give information on overall surfactant system homeostasis. Surfactant is composed of approximately 12 % protein, 80 % phospholipids and 8 % other lipids (cholesterol, triacylglycerol and free fatty acids) (Frerking 2001) and its homeostasis is regulated not only by synthesis but also by trafficking, secretion, catabolism, and recycling (Whitsett 2015). To elucidate the influence of ErbB4 and TTF-1 on more aspects of the surfactant homeostasis, studies of the metabolism cycle of surfactant proteins, genes, and other components of the surfactant system are needed.
Summary statement
Both ErbB4 and TTF-1 play critical roles in surfactant homeostasis. Our findings show a negative feedback relationship between ErbB4 and TTF-1 expression in fetal ATII cells. While there are a number of additional factors involved in the regulation of TTF-1, ErbB4, and SP-B protein expression, we here present important insights into the lung cell biology involved in the regulation of SftpB expression. More research is needed to further elucidate these interactions.
Abbreviations
- ATII cells:
-
Alveolar type II cells
- BPD:
-
Bronchopulmonary dysplasia
- RDS:
-
Respiratory distress syndrome of the neonate
- ErbB4:
-
Receptor tyrosine-protein kinase
- HER4 :
-
Human ErbB4 encoding gene
- TTF-1:
-
Thyroid transscription factor 1
- Nkx2.1 :
-
TTF-1 encoding gene
- SP-B:
-
Surfactant protein B
- SftpB :
-
SP-B encoding gene
- 4ICD:
-
ErbB4 intracellular domain
- MLE-12:
-
Mouse lung epithelium 12 cell line
- BSA:
-
Bovine serum albumin
- DMEM:
-
Dulbecco’s modified eagle medium
- FBS:
-
Fetal bovine serum
- E:
-
Embryonic day
- HBSS:
-
Hank’s buffered salt solution
- PBS:
-
Phosphate buffered saline solution
- HER4heart(−/−):
-
Homozygote ErbB4 negative transgene
- HER4heart(+/-):
-
Heterozygote transgene
- EGFP:
-
Enhanced green fluorescent protein
- HITES:
-
Hydrocortisone, insulin, transferrin, estrogen and selenium
- BCA:
-
Bicinchoninic acid
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Acknowledgements
We thank Drs. M. Gassmann and C. Birchmeier for providing the HER4heart mouse line. The studies were funded by NIH NHLBI R01 HL085648, HL037930, and Hannover Medical School.
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The authors have no conflicts of interest.
Authors’ contributions
Elger Marten - experimental design, data generation and analysis, manuscript preparation; Heber Nielsen - experimental design, manuscript preparation; Christiane Dammann - experimental design, manuscript preparation.
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Supplemental Figure 1
HER4heart transgenic mice are rescued from their lethal cardiac defect by the α-myosin heavy chain (MHC) promotor resulting in ErbB4 gene expression exclusively in the heart. (A) This cardiac rescue construct (upper blot) was detected by PCR in all mice containing at least one ErbB4-mutated allele. The mutated allele itself was detected at 320 bp and the wild type allele at 150 bp (lower blot). Representative samples of heterozygous (+/−), homozygous wild type (+/+) and homozygous ErbB4 mutated (−/−) mice are shown. (B) Representative Western blots of lung fibroblast homogenates of heterozygous (−/+) and homozygous (−/−) transgenic HER4heart mice confirmed that there is no pulmonary ErbB4 protein expression in ErbB4 −/− animals. β-actin reprobing was used to control for protein loading. (GIF 87 kb)
Supplemental Figure 2
Transfection efficiency in MLE-12 cells (A) and primary fetal ATII cells (B). Fluorescence-labeled EGFP constructs were used to identify transfection cells. The transfection efficiency was about 30–40 % in MLE-12 cells and about 20 % in primary fetal ATII cells. DAPI staining was used to identify total cell numbers. (GIF 46 kb)
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Marten, E., Nielsen, H.C. & Dammann, C.E.L. Interdependent TTF1 - ErbB4 interactions are critical for surfactant protein-B homeostasis in primary mouse lung alveolar type II cells. J. Cell Commun. Signal. 9, 207–215 (2015). https://doi.org/10.1007/s12079-015-0299-1
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DOI: https://doi.org/10.1007/s12079-015-0299-1