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Am J Physiol Lung Cell Mol Physiol 291: L191-L199, 2006. First published March 1, 2006; doi:10.1152/ajplung.00385.2005
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Hop functions downstream of Nkx2.1 and GATA6 to mediate HDAC-dependent negative regulation of pulmonary gene expression

Zhan Yin,1,* Linda Gonzales,2,* Venkatadri Kolla,2 Nibedita Rath,1 Yuzhen Zhang,1 Min Min Lu,1 Shioko Kimura,3 Philip L. Ballard,1,2 Michael F. Beers,1 Jonathan A. Epstein,1,4 and Edward E. Morrisey1,4

Departments of 1Medicine and 4Cell and Developmental Biology, University of Pennsylvania and 2Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; and 3Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Submitted 8 September 2005 ; accepted in final form 21 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hop is an unusual homeodomain protein that was first identified in the developing heart where it functions downstream of Nkx2.5 to modulate cardiac gene expression. Hop functions through interactions with histone deacetylase (HDAC) 2 to mediate repression of cardiac-specific genes, and recent studies show that HDAC activity and HDAC2 expression are decreased in people with chronic obstructive pulmonary disease. Here, we show that Hop is expressed in airway epithelium coincident with HDAC2, and expression is induced by the combination of dexamethasone and cAMP in parallel with induction of surfactant protein gene expression. Hop functions in the developing pulmonary airway, acting downstream of Nkx2.1 and GATA6, to negatively regulate surfactant protein expression. Loss of Hop expression in vivo results in defective type 2 pneumocyte development with increased surfactant production and disrupted alveolar formation. Thus Hop represents a novel regulator of pulmonary maturation that is induced by glucocorticoids to mediate functionally important HDAC-dependent negative feedback regulation.

homeodomain only protein; histone deacetylase

IMPAIRED RESPIRATORY FUNCTION resulting from immaturity of the pulmonary airways and lack of surfactant expression is among the most common causes of morbidity and mortality in premature infants. Antenatal maternal glucocorticoid administration promotes airway maturation, but premature labor does not always allow for sufficient time for this therapy to be effective, and postnatal steroid therapy is complicated by untoward neurodevelopmental effects. Hence, a more detailed understanding of the processes required for pulmonary epithelial development and surfactant expression is required to catalyze improved therapies for premature infant respiratory distress syndrome.

During late gestation, lung airways undergo a dramatic differentiation process that occurs along a proximal-distal axis to generate the distinct epithelial cell types required for surfactant protein (SP) production and gas exchange (32). This proximal-distal patterning results in distal alveolar airways that are populated by both type 2 pneumocytes, which are the cells responsible for SP production, and type 1 pneumocytes, which are the cells that generate the thin cellular interface with the capillary plexus required for efficient gas exchange. Disruption in airway differentiation results in severe neonatal respiratory distress syndromes, including bronchopulmonary dysplasia in animal models, which mimics that seen in premature infants. Adult lung disease, including chronic obstructive pulmonary disease (COPD), involves airway injury and reepithelialization, instigated by chronic injury such as cigarette smoking. The ongoing repair and remodeling process in COPD recapitulates many aspects of the differentiation programs observed in early development, including reinitiation of transcriptional programs restricted to the developing lung (2, 14). This point has been recently emphasized by studies that have implicated decreased histone deacetylase (HDAC) activity in lungs of adult patients with COPD (3, 14). HDACs function by altering chromatin structure and gene expression. Modulation of HDAC activity, by genetic or chemical means, alters embryonic development and cancer progression (19, 25, 31, 36), although specific functions in pulmonary development have not been described.

Differentiation of airway epithelium is regulated by several transcription factors, including Nkx2.1 (also known as TTF-1) and GATA6. Nkx2.1 belongs to the Nk family of homeodomain transcription factors that are known to regulate critical gene programs in the heart, lung, and other organs (7, 28). Nkx2.1 DNA binding sites are located in the regulatory regions of many lung-restricted genes, including SP genes, and loss of Nkx2.1 expression results in the loss of SP-A, -B, and -C. DNA binding sites for GATA6 are also found in many lung-restricted genes, and transgenic expression of a dominant-negative GATA6 in lung epithelium results in the loss of SP-B and SP-C (22, 35). The importance of Nkx2.1 and GATA6 is further underscored by the ability of both proteins to interact and to synergistically activate promoters of lung-restricted genes such as SP-C and Wnt7b (20, 33). The importance of Nkx2.1 in human lung homeostasis is highlighted by the recent finding of congenital pulmonary disease in patients with mutations in NKX2.1 (17).

In the heart, factors highly homologous to Nkx2.1 and GATA6 also function synergistically to regulate gene expression. Nkx2.5 and GATA4 each play important roles in cardiac development, and mutations in each factor are associated with congenital human disease (11, 13). Nkx2.5 and GATA4 form a transcriptional complex in association with serum response factor (SRF), and the activity of this complex is negatively regulated by the small homeodomain only protein, Hop (6, 30). Hop is unusual because, unlike all other known homeodomain proteins, it has divergent amino acid residues at critical positions in the third predicted {alpha}-helix that allow other homeodomain proteins to interact with DNA. Hence, Hop is unable to bind to DNA and probably mediates its negative regulatory functions by interacting with other proteins, including SRF and class 1 HDACs, in particular HDAC2 (16). Here, we show that Hop is expressed in the developing lung coincident with HDAC2. Our results demonstrate a remarkable conservation of genetic hierarchies, since we show that Hop can function as a negative regulator of Nkx and GATA gene expression in lung, as it does in the heart. We show that Hop is induced by dexamethasone in airway epithelia where it functions as an HDAC-dependent negative regulator downstream of Nkx2.1 and GATA6 and upstream of SP genes. Loss of Hop expression causes abnormal pulmonary development and type 2 pneumocyte maturation, leading to neonatal demise in the most severely affected Hop mutant neonates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hop mutant mice. The generation of Hop and Nkx2.1 mutant mice has been previously described (6, 15). Both mouse lines were kept on a C57BL/6 background for these experiments. Noon of the day that the vaginal plug was observed was considered embryonic day (E) 0.5.

Histology, in situ hybridization, immunohistochemistry, and Western blotting. Embryos younger than E16.5 were fixed for 48 h in 4% paraformaldehyde. Lung tissue from embryos E16.5 and older as well as adult lung tissue was dissected free of the embryo or adult mice and fixed in 4% paraformaldehyde for 48 h. Fixed tissues were dehydrated through a series of ethanol washes, embedded in paraffin, and sectioned (5 µm). Immunohistochemistry and in situ hybridization were performed on tissue sections as previously described (35). The HDAC2 antibody is from Zymed and was used at a 1:100 dilution, and a secondary goat anti-rabbit antibody was used at a 1:200 dilution. Details can be found at the University of Pennsylvania Molecular Cardiology Research Center web site (http://www.uphs.upenn.edu/mcrc). Western blotting was performed using the following antibodies at the indicated concentrations: Nkx2.1 1:1,000 (Santa Cruz H-190), SP-B 1:1,000 (Chemicon AB3426), Hop (1:1,000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1:2,000 (Chemicon MAB374).

Primary airway epithelial cell culture. Enriched populations of epithelial cells were isolated from second-trimester human fetal lung tissue under Institutional Review Board-approved protocols as previously described (10). After overnight culture (day 1), cells were cultured for an additional 3 days in serum-free Waymouth’s medium alone (control) or with dexamethasone (10 nM) plus 8-bromo-cAMP (0.1 mM) and isobutyl methylxanthine (0.1 mM), a combination that is referred to as DCI. Epithelial cell purity (as assessed by phosphine 3R staining) by this procedure was 86 ± 2% (n = 6), with fibroblasts as the primary contaminating cell type.

DNA microarray analysis. Total RNA was extracted from control and DCI-treated cells using RNA Stat-60 (Tel-Test), analyzed for integrity by Agilent gel technology, and converted to biotin-labeled cRNA using Affymetrix reagents and protocol (www.affymetrix.com). Biotin-labeled cRNA was hybridized to U133A Affymetrix microarray chips that contain 16–20 unique 25-mer oligonucleotide probes for ~14,500 human genes plus corresponding 12–16 probes with a single nucleotide change (missense control). U133A hybridization, washing, staining, and scanning was performed by the Stokes Research Institute Nucleic Acid Core Facility at the Children’s Hospital of Philadelphia using procedures described in the Affymetrix GeneChip Expression Analysis technical manual. Affymetrix Microarray Suite 5.0 was used to quantitate mRNA content for expressed genes. Probes for 70 control genes on each chip were used to normalize fluorescence intensity between chips, and arrays were scaled to an average intensity of 1,500 fluorescent units and analyzed independently. For the current analysis, induced genes were defined as those having a P value of <0.003 when wild-type and missense data for each probe were compared. A total of 16 chips were analyzed with cells cultured from 12 individual lungs (male and female). RNA from each preparation was analyzed by dot-blot hybridization as previously described to confirm induction of SP-B mRNA after 72 h DCI treatment (10). The degree of stimulation results are expressed as means ± SE.

Recombinant NKX2.1 adenovirus construction. Recombinant sense construct for human NKX2.1 under the CMV promoter was generated in a replication-deficient adenovirus H5.010 using the Adeno-X Expression System as per the manufacturer’s instructions (BD Biosciences, Clontech, Palo Alto, CA). NKX2.1 adenovirus plasmids were digested with Pac 1 and transfected into HEK 293 cells using Fugene 6 (Roche). Purified adenovirus was isolated, and the number of active, infective particles was determined by plaque assay. The purified adenovirus was directly used to infect cells at a concentration of 20 plaque-forming units/cell.

Quantitative PCR. Quantitative measurements of changes in gene expression were performed using an ABI 7900 real-time PCR machine and cyber-green reaction mixture according to manufacturer’s instructions and were normalized to GAPDH expression. The sequences of the oligonucleotides used in these studies are available upon request.

Small inhibitory RNA oligo inhibition of NKX2.1. Small inhibitory RNA oligonucleotides (siRNA) were synthesized and purified by Qiagen-Xeragon (Germantown, MD). Two sets of siRNAs were used that corresponded to the following sequences in the human NKX2.1 cDNA: NKX2.1A (188–208) 5'-GCACACGACUCCGUUCUCA-3' and NKX2.1D (817–837) 5'-UGAAGCGCCAGGCCAAGGA-3'. Control (nonsilencing) siRNA sequence was 5'-UUCUCCGAACGUGUCACGU-3', which bears no homology to relevant human genes. All oligonucleotides were dissolved in 100 mM potassium acetate, 30 mM HEPES-potassium hydroxide, 2 mM magnesium acetate, pH 7.4, to a final concentration of 20 µM, heated to 90°C for 60 s, and incubated at 37°C for 60 min before use to disrupt any higher aggregates formed during synthesis. Cells were cultured for 3 days in control media to deplete endogenous Nkx2.1 protein and transfected with 3–5 µg of siRNA with RNAifect transfection reagent (Qiagen) as per the manufacturer’s instructions. After 48 h, DCI was added. RNA was isolated (RNA Stat-60, Tel-Test) and analyzed by cDNA microarray analysis (6 chips) as above. Under these conditions, Nkx2.1 mRNA was inhibited 59 ± 21%, and Hop mRNA was inhibited 38 ± 12% (n = 3 lungs).

Pulmonary function testing. Lungs from wild-type and Hop mutant mice (age 3–5 mo) were lavaged, and cell counts were obtained as previously described (1). Briefly, mice were killed with pentobarbital sodium and exsanguinated by aortic transection. The trachea was exposed and cannulated. The lavage consisted of two 0.5-ml aliquots using PBS. Tidal volumes were assayed as previously described (12, 24).

Electron microscopy. Lung tissue from neonatal wild-type and Hop mutant mice (P2) were fixed in 2% gluteraldehyde with 0.1 M sodium cacodylate, pH 7.4, for 72 h at 4°C. Samples were further incubated with 2% osmium tetroxide and 0.1 M sodium cacodylate, pH 7.4, for 1 h at 4°C. Ultrathin sections were stained with lead citrate and uranyl acetate and viewed on a JEM 1010 microscope. Digital images were captured on a Hamamatsu HamC4742-95-12 charge-coupled device camera using AMT Advantage software.

Cell transfection studies. The Hop, SP-A, SP-B, and SP-C luciferase reporter plasmids have been previously described (5, 6, 9, 20). GATA6 and Nkx2.1 expression plasmids have been previously described (33). NIH 3T3 cells (2.5 x 105) in six-well tissue culture plates were transfected with the indicated plasmids using Fugene 6 as previously described using a ratio of 1:3 for DNA-Fugene (33). In the indicated experiments, trichostatin A (TSA) was added to a final concentration of 300 nM 24 h after the beginning of the transfection. All transfections were assayed after 48 h. Luciferase activity was determined using the Dual Luciferase kit (Promega). Reported values are from two different experiments performed in triplicate ± SE. Student’s t-test was used to determine statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hop and HDAC2 are expressed by airway epithelial cells. We examined Hop expression during murine pulmonary development by in situ hybridization and by examination of beta-galactosidase activity in Hop+/– embryos in which the endogenous coding sequence has been replaced with lacZ (6). Hop transcripts are first detected, albeit weakly, in the developing lung at E13.5 and are restricted to bronchial epithelium (Fig. 1A). Expression quickly increases such that, by E14.5, high levels of expression are observed throughout the conducting airways (Fig. 1B), a pattern that persists through E16.5 (Fig. 1C). Expression becomes more prominent later in gestation, encompassing both distal and proximal airway epithelium by E18.5 (Fig. 1D). Hop is known to interact with and to be influenced by HDAC2 (16). Immunohistochemistry shows that HDAC2 is expressed in both bronchial epithelium and in alveolar epithelium (Fig. 1, E and F). In situ hybridization of Hop and immunohistochemistry of HDAC2 on adjacent sections shows that genes are coexpressed in the same airways (Fig. 1, G-J). Moreover, the expression pattern of Hop and HDAC2 overlaps with that of Nkx2.1 and GATA6, both of which have been implicated in distal airway epithelial maturation (Fig. 1, K and L). Hop expression remains prominent in postnatal lung tissue (Ref. 6 and data not shown).


Figure 1
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  		Fig. 1. Expression of homeodomain only protein (Hop) during lung morphogenesis. Radioactive in situ hybridization was performed on mouse embryos at embryonic day (E) 13.5 (A), E14.5 (B and H), E16.5 (C and J), and E18.5 (D) using a riboprobe specific for Hop. Arrowheads denote Hop expression in distal airway epithelium, and asterisks denote Hop expression in proximal or bronchial airway epithelium. Histone deacetylase (HDAC) 2 expression was determined using immunohistochemistry at E14.5 (G), E16.5 (E and I), and at birth P0 (F). HDAC2 expression is observed in both columnar epithelia of the conducting airways (E and F, black arrowheads) and in alveolar epithelia (E and F, red arrowheads). Coincident expression of Hop and HDAC2 in the same airway is observed at E14.5 (G and H) and E16.5 (I and J, red arrows) using adjacent sections for immunohistochemistry and in situ hybridization. GATA6 and Nkx2.1 gene expression is observed primarily in distal airway epithelia during development (K and L).

 
HOP is induced during maturation of human airway epithelium. We have previously demonstrated that maturation of airway epithelial cells into type 2 pneumocytes and expression of SPs can be stimulated by treatment of isolated airway epithelial cells or tissue explants with DCI (10). We performed a series of microarray gene expression studies to identify genes regulated by DCI treatment in human fetal lung epithelial cells. These studies revealed a rapid (within 4 h) induction of Hop mRNA expression that closely followed activation of Nkx2.1 and preceded induction of SP-B (Fig. 2A). DCI induction of Hop expression was confirmed by Western blotting of cell extracts (Fig. 2C). Thus Hop is induced in type 2 pneumocyte differentiation in a temporal pattern similar to that of Nkx2.1.


Figure 2
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  		Fig. 2. Hop expression is induced by dexamethasone, 8-bromo cAMP, and isobutyl methylxanthine (DCI) treatment of human airway epithelial cells. A: time course of Hop induction, relative to Nkx2.1 and surfactant protein (SP)-B, was determined by cDNA microarray analysis of RNA expression. Cells from 4 lungs were cultured in the absence (control) or presence of DCI for the times shown, and then RNA for each time point was pooled to provide 10 samples (5 control, 5 DCI treated) that were analyzed by microarray. B: in separate experiments the time course of Hop, Nkx2.1, ad SP-B mRNA expression was confirmed by real-time PCR with means for 2 lungs shown. (n = 3 for Nkx2.1, *P < 0.05 vs. control). C: time course of Hop protein induction by DCI was determined by Western blot (representative blot on top), with 72 h control (no DCI) indicated by 72(–). Band intensity was determined by densitometry (Values are means ± SE of n = 3 lungs, *P < 0.05 vs. 2–24 h). Glyceraldehyde-3-phosphate (GAPDH) staining indicates equal loading of all wells.

 
Hop functions downstream of Nkx2.1. During cardiac development, Hop functions downstream of Nkx2.5, and Hop expression is significantly diminished in the hearts of Nkx2.5-deficient embryos. Nkx2.1 is closely related to Nkx2.5; therefore, we sought to determine if Hop functions downstream of Nkx2.1 in the lung. Infection of cultured human pulmonary epithelial cells with adenovirus expressing human Nkx2.1 is sufficient to induce expression of Hop (Fig. 3A). Induction of Hop expression is dose dependent and is not induced by control adenovirus expressing beta-galactosidase (Fig. 3A, and data not shown). Hop protein expression induced by Nkx2.1 approaches levels seen in DCI-treated cells. However, Nkx2.1 expression alone is not sufficient to induce expression of SP-B, although it strongly activates expression of Hop (Fig. 3, A and B). DCI, on the other hand, activates expression of Nkx2.1, Hop, and SP-B (Fig. 3A). To determine if DCI induction of Hop expression requires Nkx2.1, we utilized siRNA to knock down Nkx2.1 expression in DCI-treated cells. Using cDNA microarrays, we found that Hop mRNA expression was significantly attenuated in the presence of Nkx2.1 siRNA compared with control oligonucleotides (Fig. 3C).


Figure 3
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  		Fig. 3. Nkx2.1 expression induces Hop expression in human airway epithelial cells. A: representative Western blot shows adenoviral expression of Nkx2.1 produces a dose-dependent expression of rNkx2.1 and also induces Hop expression in a dose-dependent manner. However, SP-B expression is not induced over the dose range of rNkx2.1 expression, whereas SP-B is expressed in cells treated with DCI, which express an intermediate Nkx2.1 level. GAPDH expression (loading control) was constant with adenoviral dose. pfu, Plaque-forming units. B: quantitation of Nkx2.1-induced Hop expression compared with DCI-induced Hop expression [means ± SE of n = 4 lungs, P < 0.05 vs. control (*) and vs. Nkx2.1 (**)]. A control virus expressing green fluorescent protein (GFP; control, AdGFP) did not induce Hop expression. C: microarrays were used to assess the inhibition by Nkx2.1 small inhibitory (si) RNA treatment of DCI-treated cells on Hop expression. Nkx2.1 siRNA inhibited DCI induction (47 ± 12%) of Hop expression (P < 0.05 vs. DCI alone, n = 3).

 
If Hop expression in the lung requires Nkx2.1, we predicted that Hop expression would be downregulated in Nkx2.1-deficient lung specimens. Using in situ hybridization, we examined Hop mRNA expression in lungs of E16.5 wild-type, heterozygous, and homozygous Nkx2.1 embryos. Compared with wild-type embryos, Hop expression was diminished in heterozygous embryos and was not detected in the poorly developed lungs of Nkx2.1 null embryos (Fig. 4C). Quantitative PCR was performed to more accurately measure the change in Hop expression. These experiments show that Hop expression is reduced by >85% in Nkx2.1 null lungs (Fig. 4D). Together, these data support the concept that Hop is a direct target of Nkx2.1 in lung epithelia.


Figure 4
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  		Fig. 4. Loss of Nkx2.1 leads to loss of Hop expression in lung airway epithelium. In situ hybridization was performed on wild-type (WT; A), Nkx2.1+/– (B), and Nkx2.1–/– (C) embryos at E16.5 using a Hop riboprobe. Real-time quantitative RT-PCR was performed on cDNA samples from the same gestational age. Note severe reduction in Hop expression in Nkx2.1–/– lungs (C and D).

 
Hop is directly regulated by Nkx2.1. We sought to determine if Nkx2.1 could directly regulate transcription of Hop. We compared the ability of Nkx2.5 and Nkx2.1 to activate expression of a reporter construct composed of 5.3 kb of Hop upstream genomic sequence and a luciferase reporter gene (Fig. 5A). Both Nkx2.5 and Nkx2.1 induced ~11-fold activation of reporter gene activity (Fig. 5B). In the lung, GATA6 is coexpressed with Nkx2.1, and synergistic function has been previously demonstrated (20, 33). When cotransfected with the Hop reporter construct, GATA6 and Nkx2.1 induce >45-fold activation, consistent with synergistic activity (Fig. 5B).


Figure 5
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  		Fig. 5. Nkx2.1 and GATA6 synergistically activate the Hop promoter. A: the –5.3-kb Hop promoter has 14 GATA6 and 4 Nkx2.1 DNA binding sites, whereas the –1.3-kb Hop promoter has a single Nkx2.1 DNA binding site. B: Nkx2.5, Nkx2.1, and GATA6 (1 µg each) can individually activate the –5.3 kb Hop promoter, whereas the combination of Nkx2.1 and GATA6 (1 µg each) can synergistically activate the Hop promoter. C: Mutation of the single Nkx2.1 DNA binding site eliminates the ability of Nkx2.1 and Nkx2.5 to activate the –1.3 kb Hop promoter. Data are the average of 3 replicates from 2 assays ± SE. *P < 0.001 and **P < 0.005 vs. pcD3. Activation of the mutant Hop promoter by Nkx2.1 or Nkx2.5 is not statistically significant (P > 0.05).

 
Within the 5.3-kb genomic fragment used for these experiments, multiple Nkx and GATA binding sites are observed (Fig. 5A). However, within the proximal 1.3-kb region, only one Nkx site is identified. Consistent with this analysis, Nkx2.1 can induce activation of a 1.3-kb luciferase reporter construct. Mutation of the putative Nkx binding site abolishes Nkx2.1-induced activation (Fig. 5C). These data suggest that Nkx2.1 is a direct transcriptional activator of Hop.

Hop represses GATA6/Nkx2.1/SRF activation of lung specific genes. In cardiac tissues, GATA4 and Nkx2.5 form a transcriptional complex by interacting with SRF (6, 30). To assess whether Hop could repress GATA6/Nkx2.1/SRF activation of lung specific genes, luciferase reporter assays were performed using the –0.14-kb mouse SP-A, –0.3-kb SP-C, and –0.3-kb SP-B luciferase reporter plasmids in NIH 3T3 cells. As expected, the GATA6/Nkx2.1/SRF combination potently activated all three promoters (Fig. 6A). Coexpression of Hop resulted in dose-dependent repression of this activation (Fig. 6A). Hop did not repress control reporter plasmids, including the Renilla reporter construct used to normalize for transfection efficiency (data not shown). To determine whether HDAC activity is required for Hop-mediated repression, the transfection assays were repeated in the presence of the HDAC inhibitor TSA. TSA (300 nM) abolished Hop-mediated repression of both reporters (Fig. 6B). These data suggest that Hop negatively regulates GATA6/Nkx2.1/SRF activity in lung epithelium in an HDAC-dependent manner.


Figure 6
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  		Fig. 6. Hop represses lung-specific gene expression in an HDAC-dependent manner. A: the combination of GATA6, Nkx2.1, and serum response factor (SRF; GNS, 0.5 µg each) activates the SP-A, SP-B, and SP-C promoters (0.5 µg). Coexpression of Hop (0.5 and 2.0 µg) represses this activation in a dose-dependent manner. B: 300 nM trichostatin A (TSA) was added to transfection assays in parallel as described in A to show that Hop-mediated repression of SP-A and SP-C promoters is HDAC dependent. Data are averages of 3 replicates from 2 assays ± SE. *P < 0.005. NS, no statistical difference (P > 0.05). Difference in activation of SP-A and SP-C promoters by GATA6, Nkx2.1, and SRF in A and B is not statistically significant (P > 0.05).

 
Lung development is defective in Hop mutant mice. Based on the expression data described above, and the known function of Nkx2.1 during lung development, we examined the lungs of Hop knockout mice. Approximately 50% of Hop-deficient embryos succumb during midgestation because of cardiovascular defects (6, 30). Other Hop-deficient mice survive to birth and into adulthood. However, we noticed a significant number (~25%) of live-born Hop-deficient pups that succumbed shortly after birth or in the first postnatal days. Cardiac structure and histology appeared normal in these animals (n = 8). We examined the lungs of five Hop-deficient pups killed at P2 by routine histology, immunohistochemistry, and electron microscopy. In four of five specimens tested, we noted significant architectural abnormalities by routine histology and by electron microscopy (Fig. 7). The interairway mesenchyme of affected mutant animals was thickened compared with wild-type littermates with a coincident decrease in alveolar septal development (Fig. 7, A and B). Electron microscopy revealed dramatic increases in secreted surfactant in the airways of Hop mutant mice compared with wild-type littermates (Fig. 7, C–F). In all Hop mutants, proximal-distal epithelial differentiation was evident, as shown by appropriate patterning of SP-B, SP-C, and CC10 expression (data not shown).


Figure 7
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  		Fig. 7. Hop mutant mice have defects in lung development. Lung tissue from wild-type (A, C, and E) and Hop mutant mice (B, D, and F) at P0 was processed for both standard histology and hematoxylin and eosin staining (A and B) and for transmission electron microscopy (C-F). Hop mutant lungs exhibited hemorrhage in distal airways and increased levels of surfactant in the airways. The mesenchyme of Hop mutant lungs is also thicker than wild-type littermates.

 
Finally, we examined parameters of pulmonary function in four P21 surviving Hop-deficient mice. Presumably, the most severely affected animals are those that succumb early in postnatal life. Nevertheless, we observed 17–19% decreased lung function as measured by tidal volume and a 46–54% decrease in total cell counts from bronchoalveolar lavage in two of the four surviving adult Hop mutant mice. Decreased cell counts could reflect disrupted mesenchymal development in Hop mutant lungs. Together with the histological data showing defective alveolar development, these data indicate that Hop is a critical contributor to normal lung maturation.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this report, we show that Hop is expressed in the developing airway epithelium where it is expressed in an overlapping pattern with HDAC2. Hop expression is directly activated by Nkx2.1 and GATA6, factors that function as part of a transcriptional complex that includes SRF to activate a series of critical lung-specific genes, including those encoding surfactant A, B, and C. However, Hop expression is neither necessary nor sufficient to induce surfactant gene expression, since Nkx2.1-mediated activation of Hop also fails to induce surfactant expression, and mice lacking Hop nevertheless express surfactants. Rather, Hop functions to negatively regulate Nkx2.1/GATA6/SRF activity in a manner that requires HDAC activity.

COPD is a leading cause of adult respiratory failure caused by chronic injury. The repair process in adult patients with COPD is thought to involve an ongoing injury/repair cycle that reinitiates embryonic gene expression programs. During the injury repair process, reepithelialization occurs, leading to dysregulated type 2 pneumocyte function and concurrent changes in HDAC activity and HDAC2 expression (2, 14, 27). Our data suggest a model in which a transcriptional complex, including Nkx2.1, GATA6 and SRF, functions in both development and in the adult to mediate activation of type 2 pneumocyte gene expression, and Hop functions with HDAC2 to impart a delicate balance of negative feedback regulation. Both activation and negative feedback regulation are likely to be influenced by other genetic and environmental factors. For instance, our studies indicate that corticosteroids, such as those produced by the stress of birth or provided exogenously as medication, can induce expression of both the activator, Nkx2.1, and the repressor, Hop. Disruptions of this balance that produce either too much or too little activity of the Nkx2.1/GATA6/SRF complex result in abnormal lung development and function. Excessive Nkx2.1 and GATA6 expression can lead to decreased alveolarization and defective postnatal lung function (21, 34). Loss of function of either gene results in severe defects in lung epithelial differentiation (22, 35). Thus loss of Hop expression leads to derepression of GATA6/Nkx2.1/SRF activity, reactivating an early lung epithelial differentiation program that is deleterious to proper airway maturation or remodeling that occurs in injury or disease. The disrupted pulmonary architecture and function in Hop mutant mice support this hypothesis. Together, our data suggests that the recent observations of altered alveolar HDAC activity in COPD patients might be directly related to changes in pulmonary gene expression, at least in part, by alterations in Hop-mediated repression of transcriptional pathways that are also critical for lung development, such as those involving Nkx2.1 and GATA6.

Although HDAC activity and HDAC2 expression in adult lung have been previously reported (4, 8, 14), we believe that this report is the first to demonstrate specific expression of HDAC2 localized to pulmonary epithelial cells in the developing and mature lung, where it is strikingly coexpressed with Hop, Nkx2.1, and GATA6. Although other HDACs are expressed in the lung, our unpublished investigations indicate that none are expressed at equally high levels or in a restricted pattern similar to that of HDAC2. Hence, it will be of interest to determine if animals lacking HDAC2 in the lung, created by gene targeting, exhibit altered lung function or altered responsiveness to steroids and HDAC inhibitors. Our findings also suggest an extensive homology between gene programs operating in the heart and the lung. In developing cardiac muscle, Nkx2.5 and GATA4 function with SRF to mediate activation of critical embryonic genes (29). Mutations in either factor lead to defective cardiac development in animal models and to congenital heart disease in humans (18, 23, 26). Hop is also expressed in developing and adult cardiac muscle, where it functions downstream of Nkx2.5 to mediate negative feedback regulation of SRF-dependent gene expression (6, 30). As in the lung, this negative regulation requires HDAC activity, and HDAC inhibitors can prevent pathological cardiac hypertrophy induced by Hop overexpression (16). Hop is also expressed in other tissues, including the neural tube, blood, mammary gland, kidney, and the gastrointestinal tract, possibly indicating similar regulation of GATA and homeodomain-dependent activity in these tissues.


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These studies were supported by funding from National Heart, Lung, and Blood Institute Grants HL-075215 (to E. E. Morrisey and J. A. Epstein), HL-064632 (to E. E. Morrisey), HL-071546 (to J. A. Epstein), and HL-019737 (to P. L. Ballard and M. F. Beers). P. L. Ballard holds the Gisela and Dennis Alter Endowed Chair in Pediatrics, and J. A. Epstein holds the W. W. Smith Chair in Cardiovascular Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. E. Morrisey, Univ. of Pennsylvania, 956 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104 (e-mail: emorrise{at}mail.med.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Z. Yin and L. Gonzales contributed equally to this work. Back


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