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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Med. Author manuscript; available in PMC Apr 16, 2012.
Published in final edited form as:
PMCID: PMC3210920
NIHMSID: NIHMS313325

Thyroid Hormone Receptor Repression Linked to Type I Pneumocyte Associated Respiratory Distress Syndrome

Abstract

Although the lung is a defining feature of air-breathing animals, the pathway controlling the formation of the type I pneumocyte, the cell that mediates gas exchange, is poorly understood. In contrast, glucocorticoids and their cognate receptor (GR) have long been known to promote type II pneumocyte maturation; prenatal administration of glucocorticoids is commonly used to attenuate the severity of infant respiratory distress syndrome (RDS). Here we show that knock-in mutations of the nuclear corepressor SMRT in C57Bl6 mice (SMRTmRID) produces a novel respiratory distress syndrome due to prematurity of the type I pneumocyte. Though unresponsive to glucocorticoids, treatment with anti-thyroid hormone drugs (propylthiouracil or methimazole) completely rescues the SMRT-induced RDS, suggesting an unrecognized and essential role for the thyroid hormone receptor (TR) in lung development. We show that TR and SMRT control type I pneumocyte differentiation through Klf2, which in turn appears to directly activate the type I pneumocyte gene program. Conversely, mice without lung Klf2 lack mature type I pneumocytes and die shortly after birth, closely recapitulating the SMRTmRID phenotype. These results identify a second nuclear receptor, the TR, in type I pneumocyte differentiation and suggest a new type of therapeutic option in the treatment of glucocorticoid non-responsive RDS.

At birth the neonate undergoes a profound metabolic transition and exhibits a dramatically enhanced dependence on postnatal oxidative metabolism. While this metabolic switch is a hallmark of the fetal to newborn transition, little is known about the molecular genetics that direct this process. Physiologically, it is the first breath that expands the lung, enabling enhanced oxygenation of the blood. Failure of the lung to expand, such as in infant RDS, is one of the most common causes of neonatal mortality. The program of lung morphogenesis begins with the embryonic lung buds originating from foregut endoderm to form airways that branch into millions of alveoli required for air exchange immediately after birth1,2. These alveoli are lined by two types of epithelial cells: gas permeable type I pneumocytes which are responsible for air exchange, and type II pneumocytes which produce surfactant to reduce surface tension35.

Nuclear receptors (NRs) are ligand-activated transcription factors that play important roles in normal physiology and pathological conditions6. Their activities are not only determined by the availability of their respective ligands, but also by their association with certain coactivators and corepressors. SMRT (Silencing Mediator of Retinoid and Thyroid hormone receptors) was initially identified as a transcriptional corepressor that maintains the ‘off state’ non-liganded NRs7,8. Its interaction with NRs is mediated by two C-terminal L-X-X-X-I-X-X-X-I/L motifs termed Receptor Interacting Domains (RIDs)9,10. SMRT facilitates transcriptional repression by serving as a scaffold protein to recruit histone deacetylase complexes and chromatin remodeling factors1113.

We have generated SMRT knock-in mice (SMRTmRID) harboring point mutations within both RID1 and RID2 domains that specifically disrupt the interaction between SMRT and NRs14. Here we show that these mice on a pure C57BL6/J background die shortly after birth due to acute RDS resulting from an abnormal terminal differentiation of type I pneumocytes. Their lungs possess normal type II pneumocyte morphologies, including surfactant production, that display an unaltered gene expression program. Thus, these SMRTmRID mice provide evidence for a unique NR dependent pathway in type I pneumocyte differentiation. Unexpectedly, two anti-thyroid hormone drugs (but no other NR antagonists) restored functional type I pneumocytes, fully rescuing lung development and viability. Furthermore, we provide evidence that Klf2 is downstream of TR/SMRT signaling and is both necessary and sufficient for type I pneumocyte differentiation. Prenatal administration of glucocorticoids has long been known to promote type II pneumocyte maturation and is clinically used to mitigate the severity of infant RDS. This study identifies a second crucial NR pathway controlled by the TR, SMRT and Klf2 that, when disabled, blocks type I pneumocyte differentiation resulting in catastrophic lung collapse even in the presence of surfactant. The unexpected ability to fully rescue the syndrome with a commonly used drug suggests a potential new avenue in the management of lung prematurity and infant respiratory distress.

RESULTS

SMRTmRID mice die due to acute respiratory failure

We have previously described SMRTmRID mice harboring point mutations that specifically disrupt SMRT interactions with NRs14. Although these mice are viable in a C57BL6/Sv129 mixed background, only 14% (from a total of 361 pups) survive weaning (Supplementary Fig. 1). Viability drops to less than 1% when backcrossed to a pure C57BL6/J background (>99.5% BL6/J) (4 out of 494) (Supplementary Fig. 1). This was not due to early embryonic lethality, as all genotypes were represented at close to a Mendelian ratio at mid-gestation (E13.5, Supplementary Fig. 1).

The cause of this postnatal lethality appears to be a form of acute respiratory distress marked by severe dyspnea, cyanosis (Fig. 1a), deoxygenated blood (Fig. 1b) and diffuse lung atelectasis, as visualized by lungs that sink readily in saline (Fig. 1c). Gross evaluations showed that the lungs possessed normal orientation and lobation; there were no obvious upper respiratory airway obstructions or diaphragmatic defects. Analyses of whole body sections at different planes also revealed no histological abnormalities except in the lung (data not shown).

Figure 1
SMRTmRID mice die postnatally due to acute respiratory failure

Microscopically, both WT and SMRTmRID lungs from E18.5 to P0 mice were in the saccular stage (Fig. 1d and Supplementary Fig. 2). SMRTmRID lungs appeared partially collapsed with irregular and narrower airspaces. Alveoli were about 8 times smaller and alveolar walls were thickened, hypercellular and lined by cuboidal cells. Other pulmonary structures appeared histologically normal. No prenatal gross or histological differences were observed between SMRTmRID and WT mice at the early (E13.5) and late (E16.5) pseudoglandular stage, pointing to a late stage maturation problem (Supplementary Fig. 2 and data not shown).

SMRTmRID lungs retain premature type I pneumocytes

To ascertain the cause of postnatal death in SMRTmRID mice, we next examined their lungs under higher magnification using both light (Fig. 2a) and electron (Fig. 2b) microscopy (EM). Although type II pneumocytes exhibiting normal cuboidal morphology were readily observed, few cells with the distinctive flattened morphology typical of type I pneumocytes were found in SMRTmRID alveoli (Fig 2a). Similar observations were made at E18.5 (not shown). Under EM analysis, the SMRTmRID alveoli contained normal surfactant, lamellar bodies and tubular myelin. Alveolar walls were lined predominantly by type II pneumocytes of normal morphology and very few type I pneumocytes with the thin, gas permeable cytoplasmic extensions covering capillaries (Fig. 2b).

Figure 2
Lungs of SMRTmRID mice lack mature type I pneumocytes

While immunohistochemical examination of markers specific for both type I and II cells revealed no changes in surfactant protein levels (E18.5, Supplementary Fig. 3), dramatic reduction of typical type I pneumocyte markers (T1α and Cav1) was observed in SMRTmRID lungs (Fig. 2c). Judged by the marker staining, type I pneumocyte cytoplasmic extensions cover only 10~20% of alveolar surface in SMRTmRID mice compared to greater than 90% coverage in WT littermates. Though less dramatic, the mRNA levels of type I cell markers (T1α, Aqp5 and Cav1) were also significantly lower at both E17.5 and E18.5 (Fig. 2d and Supplementary Fig. 4), suggesting that transcription of the type I pneumocyte differentiation program is compromised at these stages. Consistent with the immunohistochemistry results, mRNA levels of type II cell marker surfactant proteins were unaffected. The mRNA levels of transcription factors NKX2.1 (also known as TTF1 or T/ebp), HNF3β and GATA6, previously implicated in lung morphogenesis1517, were similar between WT and SMRTmRID mice (Fig. 2d and Supplementary Fig. 4). Since glycogen is used in type II cells to generate surfactants, we performed PAS staining which revealed similar glycogen levels in WT and SMRTmRID lungs, further confirming that type II cells were functionally intact (Supplementary Fig. 5). Finally, as pneumocyte and alveolar development depend on normal blood flow and vascular architecture, we analyzed the terminal vascular differentiation marker PECAM-1. This revealed normal alveolar vasculogenesis within the developing alveolar structure (data not shown) which in combination with the above data suggests that type II pneumocytes, alveolar structure and vasculogenesis are normal in SMRTmRID mice.

Anti-thyroid drugs restore type I pneumocytes and rescue SMRTmRID mice

The above observations lead to the hypothesis that SMRT dependent repression of one or more NRs is required to establish the type I pneumocyte lineage and prevent acute respiratory failure. As SMRTmRID cells exhibit higher basal receptor activity as well as enhanced response to hormones such as PPARγ agonists14 and T3 (Supplementary Fig. 6), we conjectured that inhibiting NR activity might rescue the SMRTmRID phenotype.

Accordingly, several NR antagonists were tested by delivery at different doses to pregnant females at E16.5, just prior to the gestational age when the differences in gene expression and morphology were observed. Among those, antagonists of RARα (RO 41-5253), ERs (tamoxifen), and GR/PR (RU486) did not affect the survival rate of SMRTmRID pups (Supplementary Table 1). Although glucocorticoids are used clinically in infant RDS to promote surfactant production from type II pneumocytes, dexamethasone was not able to rescue SMRTmRID pups, consistent with a type I pneumocyte defect. In striking contrast, two different clinically approved anti-thyroid drugs which block iodine incorporation into thyroid hormone (TH) were observed to rescue the SMRTmRID pups (Supplementary Table 1). Both oral propylthiouracil (PTU, which also inhibits T4 to T3 conversion) and methimazole (MMI) administered through i.p. injection were able to achieve efficient rescue.

Not only are PTU/MMI-treated SMRTmRID mice born alive, but notably they show no evidence of respiratory distress, and survived to adulthood. Grossly, lungs were pink, inflated and floated in saline (Fig. 3a and 3c, compared to Fig. 1a and 1c). Their blood was found to be properly oxygenated (Fig. 3b, compared to Fig. 1b). Microscopically, both WT and SMRTmRID lungs from PTU-treated mothers were normal and in the saccular stage at E18.5 and P0 (Fig. 3d). Electron microscopy revealed terminal sacculi that were composed of normal septae with continuous cytoplasmic extension coverage from flattened type I pneumocytes as well as cuboidal type II cells containing lamellar bodies (Fig. 3e). Consistent with the morphological rescue, transcriptional activation of type I pneumocyte markers was restored and there were no significant differences in type I or II cell markers nor lung transcription factor expression between PTU-treated WT and SMRTmRID mice (Supplementary Fig. 4).

Figure 3
Anti-thyroid drugs rescue SMRTmRID animals and restore type I pneumocyte development

To confirm that PTU decreases serum TH levels in treated litters, we developed a very sensitive mass spectrometry assay to measure the concentrations of thyroid hormones T4, T3 and their metabolite reverse T3 (rT3) from small blood volumes (10~15 µl). The results showed that the serum T4 levels in both genotypes were indeed reduced after a few days of PTU treatment; serum T3 was not detectable (Supplementary Fig. 7 and Supplementary Table 2). It is notable that serum T4 levels in SMRTmRID mice were lower than in WT littermates. This is likely due to higher TR activity (as observed in SMRTmRID MEFs, Supplementary Fig. 6) in their hypothalamic-pituitary-thyroid axes, resulting in suppressed T4 production. We also note that the major embryonic form of thyroid hormone is rT3, a T4 metabolite that does not activate TR. In contrast and although thyroid hormone crosses the placenta, T4 is the dominant thyroid hormone form in pregnant females, suggesting that at this stage of embryonic development there is an active mechanism to degrade T4 to rT3 to lower TR signaling in pups (ref18 and data not shown). TR is encoded by two genes (TRα and TRβ) with distinct expression patterns; knockout animal studies have pointed to important yet different physiological functions1923. QPCR analysis of E17.5 to P0 lungs revealed that both TRα and TRβ are expressed at these stages (Supplementary Fig. 4 and data not shown), with TRβ likely to be the more abundant form (QPCR Ct values: ~25 for TRβ and ~28 for TRα).

We also tested the effect of the timing of PTU treatment on the survival rate. Notably, PTU rescued the SMRTmRID mice only when started at E16.5 and withdrawn right after birth, coinciding with dramatic lung differentiation and branching from pseudoglandular stage to saccular/alveolar stage (data not shown). Shorter periods failed to effect rescue and longer periods (starting PTU before E16.5 or withdrawing after P0) resulted in the death of all pups regardless of their genotype, presumably due to cretinism24.

SMRT regulates Klf2 and type I pneumocyte gene program in vitro and in vivo

The above results strongly indicated that TR and SMRT signaling regulate type I pneumocyte formation without affecting other cell types in developing lungs. As little is known about this cell lineage5,15, our SMRTmRID mouse provides a unique model to probe the molecular mechanisms of type I pneumocyte differentiation. As TR and SMRT do not directly bind to the promoter regions of T1α, Aqp5 and Cav1 (data not shown), we hypothesized that one or more “mediator” factors downstream of TR and SMRT must determine type I pneumocyte differentiation.

To identify such mediator proteins, we next performed genome-wide gene expression analysis of pooled WT and SMRTmRID lungs at E18.5 with or without PTU by microarray. As type I pneumocytes comprise only a small percentage of total lung cells, unsurprisingly only 21 robustly expressed genes were observed to be significantly changed between WT and SMRTmRID mice, while remaining statistically unaffected by PTU rescue at E18.5 (Supplementary Table 3). We compared expression levels by real-time PCR of these 21 candidate genes in individual littermate-controlled WT and SMRTmRID embryos at both E17.5 and E18.5 when type I markers were reduced, and found that only one gene, Klf2, also known as lung Kruppel-like factor (Lklf), was significantly different at both time points while remaining statistically unchanged with PTU treatment (Fig. 4a and data not shown). More importantly, Klf2 protein levels were almost undetectable in SMRTmRID lungs by immunohistochemistry, while strong nuclear staining of Klf2 could be clearly found in WT branching saccular epithelial cells (Fig. 4b).

Figure 4
Klf2 is reduced in SMRTmRID lungs and activates type I pneumocyte gene program

We next investigated whether TR and SMRT could repress Klf2 transcription through direct binding to its promoter or enhancers. Use of chromatin IP followed by deep sequencing (ChIP-Seq) to map genome-wide SMRT binding sites in macrophages (ref25 and data unpublished) reveals that SMRT binds strongly to a conserved enhancer region downstream of the mouse Klf2 locus (Supplementary Fig. 8). Conventional ChIP confirmed that both TR and SMRT also bind to this conserved Klf2 enhancer region (Supplementary Fig. 9) in MLE12 cells.

To determine if this enhancer contains a positive or negative TRE, we cloned it into the 3’ region of a pGL4 luciferase reporter vector. Transient transfection assays with a classical DR4 (TRE) shows, as expected, that T3 increases the DR4-Luc activity in the presence of TRβ/RXRα (Supplementary Fig. 10). However, the Klf2 enhancer is repressed by T3, identifying it as a negative or nTRE. nTREs, such as in the TSH promoter, have the property of being repressed by T3 and activated in its absence, similar to what is seen in the Klf2 gene.

Klf2 is expressed predominantly in the lung throughout embryonic and adult stages26. We next asked whether Klf2 is sufficient to induce a type I pneumocyte gene program. To this end, we overexpressed Klf2 in a mouse lung carcinoma cell line, MLE12, using adenoviral vectors. The dose of adenovirus used resulted in approximately 30~50% cell infection but did not affect cell survival or growth. Expression of type I markers, including T1α and Aqp5, was induced by ectopic Klf2 in a dose-dependent manner (Fig. 4c). Typical type II pneumocyte markers remained either unchanged (Sp-a and Sp-b) or slightly increased (Sp-c) by Klf2 overexpression (data not shown), suggesting that Klf2 specifically drove the type I cell differentiation program.

As Klf2 is a transcription factor, we next tested whether Klf2 could directly bind and activate type I cell marker gene promoters. We noticed that proximal promoters of multiple type I cell markers contain conserved Klf2 binding sites. ChIP assays demonstrated that Klf2 directly binds to the promoter regions containing these sites (Fig. 4d). Transient transfection assays showed that Klf2 directly activated the promoters of these type I markers including T1α, Aqp5 and Cav1 (Fig. 4e), while well-established type II pneumocyte transcription factor Nkx2.1 had little effect. These results suggest that Klf2 is sufficient to directly activate a type I pneumocyte gene program in vitro.

Klf2 is essential for type I pneumocyte differentiation and normal lung development in vivo

We next asked whether Klf2 is required for type I pneumocyte differentiation and normal lung development in vivo. Unfortunately Klf2 knockout mice die between E11.5 and E14.527,28, precluding the analysis of their lungs at later stages of development (E16.5~P0). To circumvent this early embryonic lethality, we generated chimeric mice using Klf2−/− ES cells to test whether Klf2 is required for normal lung development. Among 38 chimeras that were generated from 8 litters, we found two pups which showed signs of respiratory distress and died soon after delivery by C-section at E18.5. These two pups (#19 and #33) exhibited cyanosis with uninflated lungs (Fig. 5a), closely phenocopying the RDS of the SMRTmRID pups. Viable pups had no or low chimerism while the two distressed pups had highly chimeric Klf2−/− lungs (data not shown), resulting in low Klf2 mRNA and protein levels compared to their littermates (Fig. 5b and 5c). These two mice were found to have normal expression levels of Klf2 in other tissues examined (tail, liver and muscle, data not shown), suggesting that the phenotype is due to the high chimerism in the lungs but not other tissues. Other important lung transcription factors including Nkx2.1, Gata6 and Hnf3β were essentially unchanged (Supplementary Fig. 11).

Figure 5
Klf2 is essential for type I pneumocyte and normal lung development in vivo

Histological examination revealed that the two highly chimeric pups had lung atelectasis (Fig. 5d), with few flattened cells typical of type I pneumocytes, while there were abundant numbers of cuboidal type II-like cells. All type I markers were expressed at much lower levels in these two pups (Fig. 5b). Immunohistochemistry revealed a marked reduction of type I marker proteins in their lungs (Fig. 5c). The type II pneumocytes and endothelial cells were not affected as judged from SP-B, SP-C and PECAM-1 staining (Supplementary Fig. 12 and data not shown). These results demonstrate that Klf2 is essential for type I pneumocyte differentiation and normal lung development in vivo.

DISCUSSION

Embryonic lung development is orchestrated by a precisely regulated cascade of morphogens, cellular signaling molecules, hormones and transcription factors15,16,2932. Specific cell lineage determination factors have been characterized for many specialized cells of the lung17,3337. However, the factors that direct the terminal differentiation of type I pneumocytes, the cells responsible for gas exchange in the lung, have yet to be identified5,15. In this study, we delineate a novel signaling pathway in which a TR-SMRT complex is required for type I pneumocyte terminal differentiation, and provide evidence that the transcription factor Klf2 is downstream of TR signaling in normal lung morphogenesis. We show that Klf2 activates a genetic program consistent with type I—but not type II—pneumocyte development through direct interactions with type I marker promoters. Importantly, mice without lung Klf2 lack mature type I pneumocytes and die postnatally, closely recapitulating the phenotype of SMRTmRID animals. These findings identify a critical developmental role for thyroid hormone in the maturation of type I pneumocytes, and suggest that Klf2 is an essential mediator of thyroid hormone signaling in the lung.

Nuclear receptors and their coregulators play important roles in many physiological and pathological conditions. In the developing lung, the glucocorticoid receptor is well recognized for its role in promoting surfactant production. The retinoid acid receptors are critical for early lung morphogenesis and tracheal/esophageal separation38. However, the role of TR is less clear. Though some earlier reports have demonstrated that TH enhanced surfactant phospholipids synthesis from type II pneumocytes, most studies revealed that TH does not enhance surfactant protein transcription and synthesis (summarized in ref39). In supporting this, large scale human clinical trials show that administration of antenatal thyrotropin-releasing hormone in addition to surfactant supplement treatment does not provide further benefit to the neonatal outcome of preterm infants40. We show here that anti-thyroid hormone drugs could rescue the isolated type I pneumocyte defects in SMRTmRID mice. We also demonstrate that TR achieves this at least partially through direct regulation of Klf2 transcription. These results point to a critical and previously unappreciated role of the TR in type I pneumocyte development. However, it remains possible that SMRT mutation may alter the type I pneumocyte differentiation program partially through another orphan nuclear receptor or an as-yet unknown transcriptional partner, or the effects of anti-thyroid hormone drugs could be mediated though a TR-independent mechanism. It is also likely that in addition to directly regulating Klf2, the TR/SMRT complex can determine type I cell fate by affecting the proliferation and/or apoptosis of its precursors. Future work using genetic animals that have altered TR signaling in the developing lung would be instrumental to prove the essential role of TR in type I pneumocyte development.

The GR plays a critical role in lung development by acting on the immature type II pneumocyte to coordinate its maturation and the production of surfactant. It is this feature that has allowed the successful application of antenatal glucocorticoid therapy to accelerate lung development in premature infants and to ameliorate infant RDS4143. It is noticeable that approximately 20% to 30% of infants with RDS do not respond to surfactant replacement therapy and babies with lung hypoplasia and who are extremely premature (less than 24 weeks of gestation in human) do not respond well to exogenous surfactant replacement because of structural immaturity44, suggesting that overcoming lung immaturities in premature infants requires the full function of both types of pneumocytes. The rescue of type I pneumocyte immaturity in SMRTmRID mice by maternal anti-thyroid hormone drug treatment (PTU or MMI) identifies a key and unexpected role of the TR in controlling this critical transition of the fetus associated with the first breath.

In summary, we show that SMRT has a non-redundant role to NCoR and that its association with TR is critical for the terminal differentiation of the type I pneumocyte. While the GR has long been known to promote type II pneumocyte development, our findings reveal a novel TR dependent pathway for type I cell maturation and lung development (Fig 5e).

Supplementary Material

02

Acknowledgements

We thank Henry Juguilon, Malith Karunasiri, Samantha Kaufman and Yelena Dayn for technical support, Jennifer Stubbs, Christopher Kintner, Laszlo Nagy, Suk-Hyun Hong, Johan Jonker and James Fitzpatrick for helpful discussions, Marc Montminy for GFP adenovirus, Jamie Simon, Liz Grabowski and Joe Belcovson for artistic work, Estelita Ong and Sally Ganley for administrative assistance. We appreciate the help and expertise from Malcolm Wood for the EM study. We thank J. Codey and the Leona M. and Harry B. Helmsley Charitable Trust for their generous support. L.P. is a Parker B. Francis Fellow supported by the Francis Family Foundation. R.M.E. is an investigator of the Howard Hughes Medical Institute at the Salk Institute for Biological Studies and March of Dimes Chair in Molecular and Developmental Biology. This work was supported by the Howard Hughes Medical Institute and US NIH grants 2RO1DK057978, 5RO1HD027183, 5U19DK062434 (R.M.E) and RO1HL57281 (J.B.L).

Footnotes

Author Contributions

L.P. led the project, designed and performed most of the experiments. M.L. is a pathologist who evaluated all the anatomy, histology and staining results. A.A. analyzed the blood T4/rT3 levels in newborn pups using mass spectrometry. R.N. generated the SMRT knock-in mice. R.T.Y analyzed the microarray data. H-R. L. provided expertise in EM studies. G.B., J.W., D.G., M.H., K.K., M.D., H.P. and J.B.L. provided intellectual input and technical expertise. J.B.L. provided the Klf2−/− ES cells. R.M.E. supervised the project. L.P. and R.M.E. wrote the manuscript.

Competing Financial Interests

The authors declare no competing financial interests.

Accession codes

Microarray data have been deposited in the Gene Expression Omnibus (GEO) with accession code GSE30661.

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