Transgenic expression of protein phosphatase 2A regulatory subunit B56gamma disrupts distal lung differentiation

Allen D. Everett1, Craig Kamibayashi2, and David L. Brautigan3

1 Department of Pediatrics and 3 Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908; and 2 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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The distal epithelium of the developing lung exhibits high-level expression of protein phosphatase 2A (PP2A), a vital signaling enzyme. Here we report the discovery that in the lung, the PP2A regulatory subunit B56gamma is expressed in a discrete developmental period, with the highest protein levels at embryonic day (e) 17, but no detectable protein in the newborn or adult. By in situ hybridization, B56gamma was highly expressed in the distal epithelium of newly forming airways and in mesenchymal cells. In contrast, expression of B56gamma was quite low in the bronchial epithelium and vascular smooth muscle. Transgenic expression of B56gamma using the lung-specific promoter for surfactant protein C (SP-C) resulted in neonatal death. Examination of lungs from SP-C-B56gamma transgenic e18 fetuses revealed proximal airways and normal blood vessels, but the tissue was densely populated with epithelial-type cells and was devoid of normal peripheral lung structure. A component of the Wnt signaling pathway, beta -catenin, was developmentally regulated in the normal lung and was absent in lung tissue from B-56gamma transgenic fetuses. We propose that B56gamma is expressed at a particular stage of lung development to modulate PP2A action on the Wnt/beta -catenin signaling pathway during lung airway morphogenesis.

lung development; Wnt; beta -catenin; PP2A


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE LUNG FORMS AS AN OUTPOUCHING from the primitive foregut endoderm into the surrounding splanchnic mesoderm. Timely airway growth and development are orchestrated by reciprocal interactions between the epithelium and surrounding mesoderm. A complex process of interactions is involved, including cytokines, growth factors, extracellular matrix, cell membrane receptors, and transcription factors (8, 9). The signaling processes linking extracellular events to the nucleus, mediating lung growth and development, are largely unknown.

The phosphorylation of signaling molecules is key to the propagation of a growth signal. Most phosphorylation of intracellular proteins occurs on Ser and Thr residues, and the enzymes protein phosphatase (PP) 1 and PP2A account for the bulk of phosphatase activity against these targets (10, 18). PP2A is clearly necessary for normal development, because mice lacking the PP2A catalytic subunit due to homologous recombination deletion suffer early fetal demise at embryonic day (e) 6 (1). PP2A activity and level of expression are developmentally regulated in the lung (19, 21). In whole fetal lung cultures, PP2A inhibition with pharmacological inhibitors blocked branching morphogenesis and induced a G2/M cell cycle arrest (15). Despite these results, the potential functions for PP2A in the developing lung remain unclear.

Most PP2A is a heterotrimer composed of a catalytic (C) and a scaffold (A, PR65) subunit, plus one of several regulatory (B) subunits (10, 18). More than a dozen unique regulatory B subunits have been identified, and evidence indicates they confer distinctive properties to the PP2A. The B56 family of regulatory subunits is unique, because various family members show specific tissue expression, and some of the B56 family members direct PP2A to the nucleus (13, 16). To date, the expression and function of the PP2A B56 regulatory subunits in the developing lung are unknown.

The Wnt signaling pathway modulates many developmental processes (reviewed in Refs. 17, 20) and is regulated by PP2A at multiple levels (12, 14). PP2A binds axin (3), and B56 subunits can bind adenomatous polyposis coli protein (APC), with overexpression of B56 resulting in decreased levels of beta -catenin (14). beta -Catenin, a key Wnt signaling intermediate, is required for normal mammalian development. Mice null for beta -catenin die at the gastrula stage because of defects in epithelial formation (2). beta -Catenin, through its interaction with endothelial cadherin, may also play an important role in the regulation of cell migration during epithelial tubulogenesis (11), a process necessary for distal lung formation. Although these observations indicate that Wnt signaling to beta -catenin is important in development, the expression of beta -catenin in the developing lung and its regulation by PP2A are unexplored.

In the present study, we explored the expression of the PP2A regulatory subunit B56gamma . Using a combination of Western blotting and in situ hybridization, we found B56gamma to be developmentally regulated in the rat lung. Using a transgenic approach, we showed that B56gamma overexpression in the fetal lung resulted in severe alterations in fetal lung branching morphogenesis that correlated with suppression of beta -catenin levels.


    METHODS
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INTRODUCTION
METHODS
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Western blotting. Affinity-purified polyclonal antibodies were raised against the carboxy-terminal B56gamma peptide by coupling 405-EKLKEKLKMK-415 to keyhole limpet hemocyanin for immunization and using the same peptide for purification (Research Genetics, Huntsville, AL). Lungs were homogenized in ice-cold lysis buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2 µM leupeptin) with a Polytron (Brinkman Instruments, Westbury, NY). Soluble proteins were recovered by centrifugation at 10,000 g, assayed for protein concentration, frozen, and stored at -70°C. Soluble proteins (50 µg) were resolved by 10% SDS-PAGE under denaturing conditions and transferred to a nitrocellulose membrane (Trans-Blot; Bio-Rad Laboratories, Hercules, CA). Specific immunodetection with the B56gamma antibody (1:500 dilution) was by enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ) following the manufacturer's protocol. A duplicate membrane was immunoblotted with anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) as a control for loading and specific expression.

In situ hybridization. In situ hybridization was as previously described (21). Briefly, 350-bp sense and antisense digoxigenin-cRNA probes against the 5' coding region were generated from the human B56gamma cDNA. Probes were incubated with sections overnight at 37°C and washed at 42°C, and specific hybridization was detected with anti-digoxigenin- conjugated antibody plus nitro blue tetrazolium staining.

Transgenic SP-C-B56gamma mice. The full-length B56gamma human cDNA was subcloned in frame into a plasmid construct containing 3.7 kb of the human surfactant protein (SP)-C promoter and the SV40 small T intron and poly A signal (generous gift of Jeffrey Whittset) (4). Injections of the linearized transgene into the pronuclei of C57BL/6 mouse eggs and implantation into pseudopregnant host mice were performed at the University of Virginia Transgenic Mouse Facility. The genotype of embryos was determined by Southern blotting of SacI/SalI digests of genomic DNA extracted from mouse placenta. A 3.7-kb SP-C promoter fragment was [32P]dCTP labeled by random priming (Ready-to-Go Beads; Amersham Pharmacia) and hybridized at 65°C overnight. Specific hybridization was determined by phosphorimage analysis (Molecular Dynamics, Sunnyvale, CA).

Histology/Immunohistochemistry. For histological analysis, lungs were fixed at room temperature in 4% buffered paraformaldehyde for 1 h. Lungs were cryoprotected overnight in 20% sucrose and frozen in OCT medium (Miles, Elkhart, IN). Sections of 5-7 µM were cut and stained with hematoxylin and eosin for microscopic analysis. Immunohistochemical detection for beta -catenin was performed using a rabbit polyclonal antibody (Santa Cruz) at a dilution of 1:300. Controls were slides incubated with nonspecific mouse IgG (Vector Laboratories, Burlingame, CA) at the same concentration as the primary antibody. Sections were blocked for 1 h at room temperature with horse serum followed by incubation for 1 h with the primary antibody. After washing the sections for 30 min with PBS plus 0.5% Tween 20, we observed immunodetection using the avidin-biotin complex method (Vector) with an alkaline phosphatase substrate (Vector red) as a color substrate. All slides were examined and photographed on an Olympus-BX41 microscope coupled to an Olympus DP11 digital camera.


    RESULTS
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INTRODUCTION
METHODS
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DISCUSSION
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Developmental expression of PP2A subunit B56gamma . We measured the relative amounts of B56gamma regulatory subunit of PP2A in rat lung at different stages of development by Western blotting an equivalent amount of total tissue protein with a specific carboxy-terminal anti-peptide antibody (Fig. 1). The 56-kDa protein was evident at e17 and e19, but its levels dropped sharply at e20 and in the newborn to background levels. The B56gamma subunit was not detected in the adult lung, even though the A and C subunits of PP2A were abundantly expressed in this tissue (not shown). A duplicate membrane immunoblotted for actin demonstrated equivalent loading and expression of actin. We concluded that expression of the regulatory subunit B56gamma was regulated during fetal development, with a significant decrease in expression at term.


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Fig. 1.   Western blot analysis of B56gamma expression in the developing mouse lung. Soluble extracts from embryonic day (e) 17-20 and newborn (NB) lungs were prepared, and 50 µg of total protein per lane were probed with an anti-B56gamma antibody or in a duplicate membrane with anti-actin as a control.

In e17 and e19 fetal rat lung, in situ hybridization was performed to reveal the cellular distribution of B56gamma expression. As shown in Fig. 2, the highest expression of B56gamma mRNA was in the nascent peripheral airways of the developing lung. In contrast, the more proximal bronchial airways expressed B56gamma at background levels. The surrounding, less-differentiated mesenchymal cells expressed B56gamma but at considerably lower levels compared with the airway epithelium. Of interest is that the endothelium and smooth muscle of pulmonary arteries did not express B56gamma at detectable levels (Fig. 2B). The hybridizations with sense probes as controls are shown in Fig. 2, C and F, to demonstrate specificity. Thus there was narrowly restricted expression of the B56gamma subunit in cells of airway epithelium at a stage of development coincident with airway morphogenesis.


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Fig. 2.   In situ hybridization for B56gamma in e17 (A-C) and e19 (D-F) mouse lungs. Magnification in A and D, ×100; in B, C, E, and F, ×400. Antisense probes were used in A, B, D, and E to detect mRNA; in C and F, sense probes were used as controls to show specificity of hybridization. b, Bronchus; a,artery; pa, peripheral airway.

Epithelium-targeted transgenic overexpression of B56gamma . To test if the transient expression of B56gamma was critical for the process of airway morphogenesis, we overexpressed B56gamma in the fetal lung under control of the epithelium-specific promoter for SP-C. Over 200 injections were performed to produce transgenic animals, without production of a single viable founder animal. We suspected that transgenic animals were likely dying at or shortly after birth and therefore were not recovered. To examine this possibility, we performed repeat injections and killed the pseudopregnant host animals and fetuses at day 18 of gestation to examine lung morphology. We identified positive transgenic animals by genotyping the placenta by Southern blot analysis. As shown in Table 1, out of 57 fetal mice examined, seven were transgenic for SP-C-B56gamma . The gross appearance of the lungs in the transgenics was much smaller in four and modestly reduced in three, compared with nontransgenic littermates (Table 1). Lung wet weights were lower in all SP-C-B56gamma transgenics, with the greatest difference in the smallest lungs compared with nontransgenic (Table 1). Microscopic examination of fetal lung sections demonstrated a complete absence of distal lung differentiation that might account for the difference in the gross size of the lungs from B56gamma transgenic animals. As shown in Fig. 3, representative lung sections from B56gamma transgenic animals with grossly small lungs demonstrated a dense cellular lung, lacking peripheral airway formation (Fig. 3, C-F). Proximal bronchiole architecture was intact, with branching into the lung periphery, but lacked obvious budding airways. The pulmonary vasculature was readily evident, with large muscular arteries and capillaries present in the lung.

                              
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Table 1.   Surfactant protein C-B56gamma transgenic mice at e18



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Fig. 3.   Targeted transgenic expression of the protein phosphatase 2A regulatory subunit B56gamma in the lung. Immunohistochemical staining of thin sections from a transgene-negative littermate (A, B) compared with a transgene-positive littermate (C-F).

B56gamma overexpression regulates beta -catenin levels in the fetal lung. Overexpression of PP2A B56 subunits in cells in vitro results in decreased levels of beta -catenin (14). We performed immunohistochemical mapping of beta -catenin developmental expression in the e18 and adult lung. As shown in Fig. 4A, beta -catenin was present at high levels in the e18 fetal lung. All but an occasional (Fig. 4A) alveolar epithelial cell were positive for beta -catenin. Vascular endothelial cells also stained positive for beta -catenin. By contrast, the adult lung beta -catenin expression was limited to a small number of alveolar epithelial cells (Fig. 4B). At both ages, beta -catenin also was present in the adventitial layer of pulmonary blood vessels and the basement membrane of bronchi (not shown). We concluded that beta -catenin is highly expressed in the fetal lung relative to the adult. Lung sections from SP-C-B56gamma transgenic and nontransgenic littermates were stained for beta -catenin by immunohistochemistry. As shown in the representative section from the nontransgenic littermate in Fig. 5A, beta -catenin was broadly expressed in the e18 airway epithelium of the lung, with both a cytoplasmic and nuclear distribution. However, in the SP-C-B56gamma transgenic lung with severe airway defects, no beta -catenin could be detected (Fig. 5B).


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Fig. 4.   Immunohistochemical detection of beta -catenin in the e18 (A) and adult (B) mouse lung. Localization of beta -catenin is shown in red. Magnification ×400.



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Fig. 5.   Immunohistochemical staining of beta -catenin in e18 nontransgenic (A) and surfactant protein C-B56gamma (B) transgenic littermates. Sections were counterstained with hematoxylin, and localization of beta -catenin is shown in red. Arrows in A indicate nonstained epithelial cells. Magnification ×400.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The balance of phosphorylation/dephosphorylation by protein phosphatases and kinases is key to the regulation of cell proliferation, differentiation, and growth. PP2A is an abundant serine/threonine protein phosphatase present in cells as a collection of distinct holoenzymes composed of the same catalytic and structural subunits plus multiple regulatory B subunits. The B56 family of PP2A regulatory subunits includes five widely expressed paralogous genes (alpha , beta , delta , epsilon , and gamma ) (6, 7, 16).

We found that B56gamma expression is developmentally regulated in the rat lung, with expression highest in the fetal lung in contrast to barely detectable protein expression in the perinatal and adult lung. B56gamma was predominately expressed by the developing airway epithelium in the fetal lung. Because the catalytic subunit of PP2A is expressed in a similar developmental pattern in the lung (21), one can imagine that PP2A with a B56gamma subunit has a specific role in regulation of fetal lung airway development.

We demonstrate that B56gamma overexpression under control of the epithelium-specific SP-C promoter resulted in small lungs with a lack of peripheral air spaces. The importance of PP2A in normal growth and development has been shown by homologous recombination knockout of the catalytic subunit of PP2A, resulting a lack of fetal development past e5-6 (1). In the lung in particular, PP2A plays a positive role in fetal lung development because PP2A inhibitors severely impaired fetal lung growth in culture (15). The fidelity and specificity of PP2A for substrates are determined by the various regulatory subunits such as B56gamma . Thus appropriate expression of B56gamma in the developing lung is essential for normal branching morphogenesis to occur. The alteration in normal airway formation caused by B56gamma overexpression was likely not secondary to cell loss or apoptosis. The densely cellular lung seen in the B56gamma mice contrasts with the massive bleb-like spaces within the lung seen upon expression of diphtheria toxin under control of the SP-C promoter (4).

The Wingless/Wnt family of secreted glycoproteins regulates important body axis determination and patterning events (17, 20). Wnt signaling results in an accumulation of beta -catenin in the nucleus and its binding to the Lef-1/Tcf family of factors to activate transcription of target genes. At least two Wnt proteins, Wnt-5a and Wnt-11, are expressed in the developing airway epithelium (13), suggesting that Wnt signaling may be important for lung development. We found that beta -catenin was highly expressed in the e18 lung compared with the adult lung. SP-C-B56gamma transgenic overexpression resulted in suppression of beta -catenin levels during this period of development. Similarly, B56 overexpression in cultured cells resulted in phosphorylation-induced proteosomal degradation of beta -catenin, likely through increased activation of glycogen synthase kinase-3 (14). Importantly, B56 expression in cultured cell lines also decreased downstream transcriptional activation of at least the Lef-1/Tcf1 target gene siamois (5). Together these observations indicate that B56 subunits target PP2A and thereby promote degradation of beta -catenin. PP2A can also play a positive role in regulating Wnt signaling (12). With the use of microinjection assays in Xenopus laevis, the catalytic subunit of PP2A (PP2A-C) was found to potentiate Wnt signaling, whereas a B56 subunit inhibited signaling. These observations suggest that different B regulatory subunits of PP2A can redirect the phosphatase to provide both positive and negative input into the Wnt signaling cascade.

In summary, the developmentally expressed PP2A regulatory subunit B56gamma dramatically alters lung branching morphogenesis. When B56gamma was expressed as a tissue-specific transgene, it suppressed beta -catenin levels. These data indicate an important role for PP2A in the regulation of lung growth and development, possibly through regulation of the Wnt signaling pathway.


    ACKNOWLEDGEMENTS

A. D. Everett was partially supported by the University of Virginia Children's Medical Center and the Virginia Thoracic Society. D. L. Brautigan was supported by United States Public Health Service Grant CA-77584, and C. Kamibayashi by American Heart Association, Texas affiliate Grant 95R-091.


    FOOTNOTES

Address for reprint requests and other correspondence: A. D. Everett, Univ. of Virginia Health System, Pediatric Cardiology, MR4 Bldg., PO Box 801356, Charlottesville, VA 22908-1356 (E-mail: ade5r{at}virginia.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.

10.1152/ajplung.00262.2001

Received 16 July 2001; accepted in final form 15 January 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 282(6):L1266-L1271
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