Upregulation of erythropoietin receptor during postnatal and postpneumonectomy lung growth
David J. Foster,1
Orson W. Moe,1,2 and
Connie C. W. Hsia1
1Department of Internal Medicine, University of Texas Southwestern Medical Center and 2Medical Service, Department of Veterans Affairs Medical Center, Dallas, Texas 75390-9034
Submitted 29 March 2004
; accepted in final form 27 July 2004
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ABSTRACT
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Circulating erythropoietin (EPO) stimulates erythrocytosis, whereas organ-specific local EPO receptor (EPOR) expression has been linked to angiogenesis, tissue growth, and development. On the basis of the observation of concurrent enhancement of lung growth and erythrocyte production during exposure to chronic hypoxia, we hypothesized that a paracrine EPO system is involved in mediating lung growth. We analyzed EPOR protein expression in normal dog lung tissue during postnatal maturation and during compensatory lung growth after right pneumonectomy (PNX). Membrane-bound EPOR was significantly more abundant in the immature lung compared with mature lung and in the remaining lung 3 wk after PNX compared with matched sham controls. COOH-terminal cytosolic EPOR peptides, which were even more abundant than membrane-bound EPOR, were also upregulated in immature lung but differentially processed after PNX. Apoptosis was enhanced during both types of lung growth in direct relationship to cellular proliferation and EPOR expression. We conclude that both developmental and compensatory lung growth involve paracrine EPO signaling with parallel upregulation but differential processing of EPOR.
dog; postnatal development and maturation; compensatory lung growth; apoptosis; immunohistochemistry; immunoblot
IT IS NOW RECOGNIZED that erythropoietin (EPO) subserves both endocrine and paracrine functions. Circulating EPO produced by adult kidney and liver (22) stimulates erythrocytosis by inhibiting apoptosis of erythroid progenitors (28). Loss of EPO production in null transgenic mice results in failure of erythropoiesis and is lethal by embryonic day 13.5 (57). In addition to endocrine release, localized production of EPO in extrahematopoietic tissues exerts organ-specific paracrine effects. In brain, EPO protects neurons from hypoxia and ischemic injury (43, 49). EPO stimulates proliferation and migration of endothelial cells (2), and uterine EPO production mediates angiogenesis during the estrus cycle (58). EPO null mice develop ventricular hypoplasia, suggesting a role for EPO in cardiac morphogenesis (56). These effects are mediated by EPO receptors (EPOR) that are widely expressed in neural and endothelial cell lines (3, 36) and in many tissues including lung (27). EPOR expression is developmentally regulated in mouse brain with significantly higher mRNA levels in prenatal animals (32, 33). Expression of EPOR has been documented in fetal lung (27) but not postnatal lung. Low levels of EPO mRNA are present in lung (15), but the function of a paracrine EPO axis in the lung is unknown.
Circulating EPO is markedly stimulated by hypoxia and anemia (45, 52), and local production of EPO is increased by hypoxia in brain and uterus (8). EPO and EPOR are abundantly expressed in vasculature and hypoxic regions of breast cancer tissue (1). Exogenous EPO is as effective as vascular endothelial growth factor (VEGF) in promoting in vitro capillary outgrowth (23). Thus EPO stimulates at least two critical tandem processes to enhance oxygen transport under hypoxic conditions, erythropoiesis and angiogenesis. We theorized that EPO could also exert direct effects on the lung. Sustained alveolar hypoxia accelerates normal postnatal lung growth in rodents (4, 7, 10, 31) and dogs (25) and enhances compensatory lung growth in pneumonectomized rats (46). The concurrent enhancement of alveolar growth, erythropoiesis, and angiogenesis by chronic hypoxia suggests a common regulatory pathway in which autocrine/paracrine EPO signaling mediates lung growth, just as circulating EPO mediates red blood cell production. As a first step toward testing this hypothesis, we characterized EPOR protein expression in canine lung using two models of lung growth, postnatal maturation (3 and 12 mo of age) and postpneumonectomy (PNX) compensatory growth. Because EPO is known to inhibit apoptosis (28, 49), we related EPOR content to apoptotic activity in the lung in both models.
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MATERIALS AND METHODS
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Animal procedures and tissue collection.
These studies were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center. To examine postnatal lung growth, lung tissue was obtained during thoracotomy from peripheral and central locations within the right upper lobe of normal foxhounds at 3 or 12 mo of age (n = 6 per group). To examine post-PNX lung growth, litter- and sex-matched immature foxhounds underwent either right PNX or thoracotomy without PNX (sham) at 9 wk of age. Under isoflurane anesthesia, the right lung was exposed via a lateral thoracotomy through the fifth intercostal space. The lobar arteries and veins were individually ligated and cut between ligatures. The right main stem bronchus was stapled and cut. The stump was immersed under saline to check for leaks. After hemostasis was ensured, the thorax was closed in five layers. Residual air within the thorax was aspirated. Sham animals underwent right thoracotomy without lung resection. At 3 wk (n = 6 per group) or 10 mo (n = 5 per group) following surgery, corresponding to
3 and 12 mo of age, respectively, animals were deeply anesthetized with intravenous pentobarbital and mechanically ventilated. Via a left thoracotomy, the left upper lobe was removed. Lung tissue samples were taken from the peripheral and central regions of the lobe, rinsed with saline, flash-frozen in liquid nitrogen, and stored at 70°C for later protein analysis. Additional tissue samples were fixed in formalin or Hollande's fixative for 24 h, washed with 70% ethanol, and embedded in paraffin for immunohistochemical analysis.
Immunoassays.
Lung tissue (50100 mg wet weight) was minced on ice and homogenized by polytron in buffer containing 300 mM sucrose, 20 mM Tris, pH 8.0, 10 mM HEPES, 5 mM EGTA, 2 mM 2-mercaptoethanol, and protease inhibitors aprotinin (5 µg/ml), pepstatin A (1.5 µM), leupeptin (100 µM), trypsin inhibitor (5 µg/ml), p-aminobenzamidine (200 µM), and PMSF (1 mM). The homogenates were cleared by centrifugation at 15,000 g, and the supernatant was ultracentrifuged at 150,000 g to separate membrane-bound and soluble cytosolic protein. Membrane pellets were resuspended in buffer containing 5 mM Tris, pH 6.8, 10% glycerol, 1% 2-mercaptoethanol, and 1% SDS. Total protein in the tissue lysates and membrane-bound and cytosolic protein fractions was quantified by Bradford assay (Bio-Rad Laboratories, Hercules, CA).
EPOR expression was quantified from replicate immunoblots comparing immature and PNX dogs to their respective control groups (mature or sham). Separate immunoblots were prepared for membrane and cytosolic protein extracts. Equal quantities of protein from each animal in each group were resolved on denaturing polyacrylamide gels and blotted onto polyvinylidene difluoride membranes. Blots were blocked in Blotto-Tween (5% nonfat dry milk, 0.05% Tween in PBS) for 1 h and incubated with primary antibodies in Blotto-Tween for a minimum of 2 h. Antisera used were rabbit polyclonal anti-EPOR antibody M-20 (raised against a peptide antigen located within 50 amino acids of the COOH terminus of mouse EPOR, 1 µg/ml) and H-194 (raised against human EPOR, NH2-terminal amino acids 21214, 2 µg/ml), both from Santa Cruz Biotechnology, Santa Cruz, CA. Immunolabeling was visualized by chemiluminescence (Western Lightening; Perkin Elmer Life Sciences, Boston, MA) and quantified by densitometry. At least three immunoblots were prepared comparing immature and mature dogs or sham and PNX dogs at each time point. Each assay utilized independent tissue samples obtained from different locations in each dog lung. Specificity of the antiserum reactions was established in separate blocking experiments using either 5x excess blocking peptide for M-20 antiserum (Santa Cruz Biotechnology) or 50x excess recombinant human soluble EPOR (R&D Systems, Minneapolis, MN) for H-194 antiserum.
The relative abundance of membrane and cytosolic EPOR was estimated in three immature and three mature lungs by immunoblot. In this case, proteins from both membrane and cytosolic extracts were resolved on the same denaturing polyacrylamide gel. A constant amount (4 µg) of each membrane fraction protein was loaded, and loading of the corresponding cytosolic protein was proportional to the ratio of total cytosolic to membrane protein in each lysate, which varied between 9:1 and 20:1. The signal intensities of EPOR in each fraction were thus proportional to their relative abundance in total lung lysate.
Immunohistochemistry.
Paraffin-embedded blocks were sectioned at 4-µm thickness. Sections were pretreated in 0.05% saponin for 30 min and immunostained with M-20 antiserum at 0.125 µg/ml in the presence and absence of 10x blocking peptide. Normal rabbit IgG at the same concentration was used as a negative control. Bound primary antibody was detected using the rabbit Unitect ABC kit (Oncogene Research Products, San Diego, CA), and sections were counterstained with hematoxylin. For colocalization experiments, paraffin-embedded blocks were sectioned at 2-µm thickness, and adjacent sections were mounted on separate slides. One section was immunostained with M-20 antiserum as described above; the adjacent section was immunostained with one of the following antisera: rat anti-human procollagen I NH2-terminal monoclonal antibody (Chemicon International, Temecula, CA); mouse anti-human
-smooth muscle actin monoclonal antibody (Chemicon International); rabbit anti-human von Willebrand factor polyclonal antibody (DakoCytomation, Carpinteria, CA); or mouse anti-human monocytes/macrophages monoclonal antibody (Chemicon International). Antigen unmasking was performed according to manufacturer's instructions, and sections were counterstained with hematoxylin. Colocalization of EPOR staining with these cell markers was examined for at least 100 EPOR-expressing cells in adjacent lung sections from four animals.
TdT-mediated dUTP nick end labeling assay.
The protocol for TdT-mediated dUTP nick end labeling (TUNEL) was generously provided by Dr. Steven Kernie, Department of Pediatrics, University of Texas Southwestern Medical Center. Assays were performed on four tissue sections (4 µm) per lung; two sections were fixed in formalin and two in Hollande's fixative. Sections were digested in proteinase K (10 µg/ml). Endogenous peroxide was blocked by incubation in 3% H2O2. Sections were incubated for 60 min at 37°C with 40 µM biotin-112'-deoxyuridine-5'-triphosphate (Enzo Life Sciences, Farmingdale, NY) and 0.3 U/µl TdT (Invitrogen, Carlsbad, CA). Labeled apoptotic cells were visualized using Vectastain Elite ABC-Peroxidase kit (Vector Laboratories, Burlingame, CA) and hematoxylin counterstain.
Data analysis.
EPOR abundance was assessed from replicate immunoblots comparing immature and mature dogs or sham and PNX dogs at each time point. Signal intensities on each blot were expressed as a percentage of the mean signal intensity of the respective control group (mature or sham) on the same blot. The relative signal intensities from at least three replicate assays were combined and compared by unpaired t-test (n = 15 or 18 independent observations per group). A P value <0.05 was considered significant.
TUNEL-positive cells were counted in 50 nonoverlapping serial images taken from each section (
250x), beginning from a random start. To estimate total cell nuclei, the images were converted to grayscale, thresholded, and subjected to particle analysis using the public domain ImageJ software (developed at the National Institutes of Health and available at http://rsb.info.nih.gov/ij). The apoptotic index of TUNEL-positive nuclei was calculated as a percentage of the total number of nuclei in the section, i.e., = positive nuclei/total nuclei and expressed as a fraction of the mean apoptotic index for the respective controls (mature or sham) in the same assay to normalize for interassay variation. Comparisons between groups were made by unpaired t-tests (n = 2024 sections per group).
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RESULTS
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Characterization of EPOR expression.
Figure 1 shows immunolocalization of EPOR expression in immature dog lung using a polyclonal antibody against a COOH-terminal epitope. Labeling localized to discrete cells scattered throughout alveolar septa, predominantly within interstitium. Labeling was primarily perinuclear and punctuate in appearance (Fig. 1A). Additional staining was observed along the apical surface of airway epithelium (Fig. 1B). Immunolabeling was abolished when the antiserum was preincubated with blocking peptide (not shown).

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Fig. 1. Representative photomicrographs of erythropoietin receptor (EPOR) immunolabeling in airway and parenchyma of immersion-fixed dog lung. Insets in A and B show higher magnification of area designated by arrow.
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To determine the cell type expressing EPOR, adjacent sections were immunostained for EPOR and markers of common lung cell types, including fibroblasts (procollagen I), myofibroblasts (
-smooth muscle actin), endothelial cells (von Willebrand factor), and alveolar macrophages (anti-human monocytes/macrophages monoclonal antibody). Approximately 1020% of EPOR-expressing cells colocalized with the monocyte/macrophage marker (Fig. 2). Thus EPOR is expressed in at least a subset of alveolar macrophages.

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Fig. 2. Representative photomicrograph of EPOR colocalization with monocyte/macrophage marker. Insets show higher magnification of cells indicated by arrows. A: EPOR immunolabeling. B: immunolabeling of adjacent section by anti-human monocytes/macrophages monoclonal antibody.
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Immunoblot of lung homogenate using COOH-terminal antiserum revealed three prominent bands (
130, 50, and 37 kDa; Fig. 3A). The 130-kDa protein localized to the membrane and approximated the expected size of dimerized EPOR, based on the length and glycosylation of cloned human EPOR (59). The smaller bands (
50 and 37 kDa) localized to the cytosol. Immunostaining of all three bands was blocked in the presence of 5x excess concentration of the peptide epitope, indicating specific protein-antibody interactions.

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Fig. 3. Immunoblot of EPOR expression in immature lung. A: detected with COOH-terminal antibody (M-20) in whole lung lysate (L) and after fractionation into membrane-bound (M) and cytosolic (C) extracts ± 5x blocking peptide. One specific band was detected in the membrane fraction and 2 specific bands in the cytosolic fraction. B: detected with NH2-terminal antibody (H-194) ± 50x recombinant human soluble EPOR. One specific band was detected in the membrane fraction and 1 specific band in the cytosolic fraction.
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To further examine protein fractions retained in the cytoplasm, additional blots were immunostained with polyclonal antiserum raised against an NH2-terminal epitope of human EPOR (Fig. 3B). Because a blocking peptide was not available for this antibody, blocking experiments were performed using 50x excess concentration of recombinant human soluble EPOR. The 50- and 37-kDa proteins were not detected by the NH2-terminal antibody, suggesting that they are COOH-terminal EPOR fragments. Instead, the antiserum immunostained a 150-kDa protein in the membrane and a protein
30 kDa in the cytosol; both were blocked in the presence of excess soluble EPOR. The 30-kDa protein corresponds to a soluble form of the receptor that has been described in mouse and human (38, 39). An
65-kDa nonspecific band was also labeled. It is unclear why the NH2-terminal antiserum recognized a slightly larger membrane-bound protein than did the COOH-terminal antibody.
EPOR protein during developmental lung growth.
The relative abundance of membrane and cytosolic EPOR proteins was examined in three immature and three mature dog lungs using the COOH-terminal antiserum (Fig. 4A). To appreciate the relative distribution, a constant amount of membrane protein was loaded, whereas cytosolic protein loaded on the same gel was proportional to the ratio of total cytosolic to membrane protein in the lung lysates. Signal intensities for each protein were averaged per group and expressed as a percentage of the 130-kDa protein in membrane of mature lung (Fig. 4B). Expression of the membrane-bound 130-kDa protein was approximately twice as high in immature lung as in mature lung. Levels of the cytosolic 50- and 37-kDa peptides greatly exceeded the amount of 130-kDa protein and represented 89% of total immunoreactive EPOR in immature lung and 83% in mature lung. Abundance of the 50-kDa peptide in immature lungs was 4.5-fold that of mature lungs, whereas abundance of the 37-kDa peptide in immature lungs was 1.8-fold that of mature lungs (Fig. 4B). Thus abundance of the cytosolic EPOR peptides decreased to a greater extent than the membrane-bound receptor during postnatal lung maturation.

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Fig. 4. To show the relative abundance of EPOR COOH-terminal peptides in membrane and cytosolic fractions from immature and mature lung, 4 µg of membrane protein from 3 immature and 3 mature lungs were loaded. Cytosolic protein loading (63, 78, 37, 77, 44, and 59 µg for cytosolic lanes 712, respectively) was proportional to the ratio of total cytosolic-to-membrane protein in the starting lysates. A: blot immunostained with COOH-terminal antibody. B: signal intensities in A were averaged per group per protein and expressed as a percentage of the mean (±SE) in the membrane fraction of mature lungs.
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Figure 5 shows results from replicate immunoblots that differ from Fig. 4 in that equal quantities of membrane or cytosolic proteins were loaded on separate gels. In immature (3-mo-old) lung, membrane-bound EPOR detected using COOH-terminal antiserum was enhanced to 160% of that in control (mature) lung (P < 0.01, Fig. 5A); the 50- and 37-kDa cytosolic EPOR peptides were increased to 707% (P < 0.001) and 266% (P < 0.01) of mature level, respectively. EPOR protein labeled using NH2-terminal antiserum was also higher in immature lung (Fig. 5B); membrane-bound EPOR was 167% (P < 0.01) and soluble EPOR was 223% (P < 0.001) that in mature lung. Thus two different antisera showed a consistently greater abundance of EPOR in immature lung and differential regulation of EPOR peptides during postnatal development.

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Fig. 5. EPOR expression in immature and mature lung (means ± SE). Replicate immunoblots were performed using equal quantities of protein from both membrane and cytosolic extracts. Representative immunoblots are shown for 2 animals per group. Signal intensity, normalized to 20 µg of protein loaded, is expressed as a percentage of the mean intensity obtained in the mature group in each of triplicate assays (n = 6 animals per group; total of 18 independent observations). A: COOH-terminal antibody. B: NH2-terminal antibody. P values indicate immature vs. mature.
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EPOR protein during compensatory lung growth.
Three weeks after PNX, membrane-bound EPOR immunostained with COOH-terminal antiserum increased to 169% of sham level (P < 0.05, Fig. 6A) and then returned to control level by 10 mo after surgery (Fig. 6C). A similar pattern was observed using NH2-terminal antiserum (Fig. 6B); membrane-bound EPOR increased to 223% of sham level (P < 0.01) 3 wk post-PNX and was similar to sham by 10 mo (Fig. 6D). Thus expression of membrane-bound EPOR increased further early after PNX, similar to that observed during postnatal maturation. In contrast, expression of the 50-kDa cytosolic COOH-terminal peptide, so markedly elevated in immature lung, was unchanged 3 wk after PNX (Fig. 6A) and, in fact, fell to 62% of sham level 10 mo post-PNX (P < 0.01, Fig. 6C). The 37-kDa cytosolic EPOR fragment was modestly elevated by 35% early after PNX (P < 0.001, Fig. 6A) and returned to sham level 10 mo post-PNX (Fig. 6C). Cytosolic EPOR immunostained with NH2-terminal antiserum showed no change at either time point after PNX. Results indicate higher expression of membrane-bound EPOR during post-PNX lung growth in immature dogs, accompanied by alterations in intracellular processing of the receptor.

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Fig. 6. EPOR expression after pneumonectomy (PNX; means ± SE). Representative immunoblots are shown. Signal intensity, normalized to 20 µg of protein loaded, is expressed as a percentage of the mean intensity obtained in the sham group in each of triplicate assays (1518 independent observations per group). A and B: 3 wk post-PNX, n = 6 animals per group. C and D: 10 mo post-PNX, n = 5 animals per group. A and C: COOH-terminal antibody. B and D: NH2-terminal antibody. P values indicate PNX vs. sham.
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Apoptosis.
In immature lung, apoptosis index was 466% that in mature lung (P < 0.05, Fig. 7A). At 3 wk after PNX, apoptosis index was 153% that in sham lungs (P < 0.05, Fig. 7B) but fell to 59% of sham levels by 10 mo post-PNX (P < 0.001, Fig. 7B). Thus apoptotic activity directly correlates with EPOR during postnatal and compensatory lung growth. Representative images showing TUNEL staining in immature and mature lung are shown in Fig. 7, C and D.

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Fig. 7. Apoptotic index (means ± SE) estimated in 4 independent sections per lung and 50 serial images per section (250x) are expressed as a percentage of the mean value in the respective control group (mature or sham) in each assay. A: postnatal growth, n = 6 animals per group. B: post-PNX, n = 6 animals per group 3 wk after surgery and n = 5 animals per group 10 mo after surgery. P values indicate comparison to the respective control groups (mature or sham). C and D: representative photomicrographs of TdT-mediated dUTP nick end labeling staining in immature (C) and mature (D) lung.
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DISCUSSION
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Summary of results.
We report the first characterization of EPOR expression and regulation in the lung during postnatal maturation and post-PNX compensatory lung growth. Results are summarized in Fig. 8. Membrane-bound EPOR was higher in immature compared with mature lung but constituted only a small fraction of total EPOR. Cytosolic COOH-terminal EPOR peptides were present at up to eightfold greater abundance than membrane-bound EPOR and were increased in immature lung, as was an NH2-terminal soluble EPOR protein. Compensatory lung growth 3 wk post-PNX was associated with significantly elevated membrane-bound EPOR that subsequently declined to sham level 10 mo post-PNX. In contrast, cytosolic EPOR fragments were either unchanged or only modestly higher 3 wk post-PNX, with the 50-kDa cytosolic peptide significantly lower than sham levels by 10 mo post-PNX. Thus expression of membrane EPOR correlates with states of active cellular proliferation, but expression of soluble EPOR peptides appears to be differentially regulated depending on the type of growth. Apoptotic activity was higher in immature compared with mature lung and also higher 3 wk after PNX but eventually fell below sham level by 10 mo. Higher EPOR expression correlates with heightened apoptosis during both postnatal and compensatory lung growth.
Characterization of EPOR expression.
Human and murine EPOR cDNAs (11, 21, 26, 55) each encode a transmembrane glycoprotein that shares common motifs with the cytokine receptor superfamily (60). The murine EPOR cDNA coding region translates to 507 amino acids and generates a 66-kDa protein after acquisition of oligosaccharide in the Golgi (59). An alternatively spliced EPOR transcript lacks the transmembrane domain, resulting in a soluble form of the receptor that retains ligand binding activity (38, 39). Processing of EPOR is unusual in that the majority of newly synthesized molecules are degraded without reaching the cell surface (29, 37, 53, 59). Endoproteolytic cleavage of murine EPOR in a post-Golgi compartment results in COOH-terminal fragments of 46 and 39 kDa (40). Interaction of EPOR with Janus kinase 2 in the endoplasmic reticulum controls cell surface expression (20). Binding of a single EPO molecule results in either dimerization of EPOR monomers (54) or conformational changes in preformed EPOR homodimers (34, 42).
Juul et al. (27) reported EPOR immunoreactivity in human fetal lung tissue beginning at 6 wk postconception, localized to round interstitial cells, at least some of which were macrophages. Immunoreactivity increased 18 wk postconception and included bronchial epithelial cells and smooth muscle cells of bronchial arteries. These reports are consistent with our data localizing EPOR in postnatal lung to discrete interstitial cells scattered throughout lung parenchyma and also in airway epithelium, of which 1020% colocalized with a monocyte/macrophage marker. The identity of other EPOR-expressing cells is still unclear, since no colocalization was observed with markers for fibroblasts, myofibroblasts, and endothelial cells. The perinuclear and punctuate labeling pattern we observed for EPOR is similar to that reported in transfected Ba/F3 and NIH/3T3 cells, where EPOR immunoreactivity colocalized with a lysosomal marker, suggesting the possibility of receptor degradation (40, 41).
Immunoblot of lung lysates using antisera raised against COOH-terminal and NH2-terminal epitopes specifically recognized EPOR proteins that localized to either membrane-bound or cytosolic fractions. Both antisera detected a single protein in membrane preparations, although the NH2-terminal antiserum recognized a slightly slower migrating protein (150 kDa) than did COOH-terminal antiserum (130 kDa). The reason for this difference is not clear. We were somewhat surprised by the lack of EPOR monomers,
66 kDa after glycosylation in mouse and human (55, 59). There is crystallographic evidence (34) as well as fragment complementation assays (42) to support the notion that EPORs exist on the plasma membrane as preformed dimers. The relatively mild denaturation conditions we used may have left most of the receptor in its dimerized form.
COOH-terminal antiserum labeled EPOR-reactive peptides of
50 and 37 kDa that were not recognized by NH2-terminal antiserum. The mobility of these fragments is similar to the 46- and 39-kDa COOH-terminal fragments reported by Neumann et al. (40) in murine Ba/F3 cells transfected with murine EPOR cDNA. In Ba/F3 cells, these intermediates constituted a minor percentage of newly synthesized EPOR and resulted from endoproteolytic cleavage in the exoplasmic region of EPOR, probably in a late or post-Golgi compartment; however, they were membrane anchored and eventually degraded in lysosomes along with intact EPOR (40). In contrast, in dog lung, the 50- and 37-kDa COOH-terminal peptides are in the cytosol. Their combined abundance greatly exceeded that of membrane EPOR by eightfold in immature lung and fivefold in mature lung. It is conceivable that they are not degradation products but instead are functional. Neither peptide was detected using antibody raised against an NH2-terminal sequence containing the EPO binding site; thus they may lack some or all of the ligand binding domain. However, both peptides likely retain docking sites in their COOH-terminal cytoplasmic domains for intracellular signaling mediators that could activate downstream signal transduction in an EPO-independent manner. An alternatively spliced EPOR transcript has been identified in human brain that lacks signal peptide and NH2-terminal sequence and could potentially be translated into truncated EPOR restricted to the cytoplasm (9). The cytoplasmic EPOR peptides we observed may have a similar origin.
Postnatal lung maturation.
Null mutations in the EPOR gene (57) result in the failure of fetal liver erythropoiesis and embryonic lethality. Juul et al. (27) have shown a wide distribution of EPOR in human fetal myocardium, lung, retina, and kidney. EPOR level is high in embryonic mouse brain (33) and recedes to nondetectable levels before birth. Here we report for the first time a 6070% greater abundance of membrane EPOR in the postnatal growing lung relative to the adult lung. The higher EPOR expression directly correlates with vigorous cell proliferation and higher levels of EGF and EGF receptor previously reported in these same animals (16). Importantly, relative expression of cytosolic EPOR peptides was elevated to an even greater extent than membrane EPOR in immature compared with mature lung (
7.1-fold and 2.7-fold for the 50- and 37-kDa peptides, respectively.) These cytoplasmic peptides may be functionally active in regulating postnatal lung maturation.
Compensatory lung growth.
Increased mechanical strain imposed on the remaining lung post-PNX is the predominant signal that initiates and sustains compensatory alveolar growth, but alveolar hypoxia may also play a role in stimulating EPOR expression in the lung. Alveolar-arterial oxygen tension gradient is normal at rest following PNX but becomes abnormally elevated upon exercise (19). The effect of hypoxia on lung growth is age and species dependent. In rodents, hypoxia depresses alveolar formation in the perinatal period (35) but has a stimulatory effect in older animals (5). Hypoxia stimulates alveolar growth in 2- to 3-mo-old dogs but has no effect in adult dogs (24, 25). In some studies, severe hypoxia blunts somatic growth and precludes an absolute increase in lung weight or volume above control lungs even though lung volume per kilogram of body weight is higher. However, even in studies utilizing moderate hypoxia where body weight is not depressed, hypoxia still significantly enhances alveolar growth in growing animals (24, 25).
In immature animals, compensatory alveolar growth following PNX is vigorous (51), consisting of early cellular proliferation (16) followed by tissue remodeling. The process superficially parallels postnatal lung maturation, although distinct differences exist in specific markers of cellular growth and differentiation (e.g., surfactant-associated protein A and EGF and its receptors) between these two models. Membrane EPOR content in the remaining lung was higher 3 wk post-PNX and returned to sham levels by 10 mo post-PNX. The magnitude of the increase 3 wk post-PNX (to 223% of sham level) is similar to that observed in immature animals relative to mature controls, suggesting that membrane-bound EPOR subserves similar functions during postnatal and compensatory lung growth.
In contrast to the uniform elevation of all cytosolic EPOR peptides seen during maturation, by 3 wk after PNX only the cytosolic 37-kDa COOH-terminal peptide was selectively and modestly increased (by 35%) above the already elevated level seen in the immature lung. There was no change in the levels of other two soluble EPOR peptides after PNX, suggesting that compensatory alveolar growth involves different regulation of protein-processing pathways of EPOR than developmental alveolar growth. By 10 mo post-PNX, levels of COOH-terminal peptides in the remaining lung were lower than in similarly aged mature lungs, suggesting altered EPOR synthesis and/or differential processing of soluble EPOR peptides during post-PNX remodeling. We do not know how local EPO concentration changes during lung growth or its relationship to EPOR expression. Erythroid progenitor cell lines with elevated sensitivity to EPO have been identified that do not differ in the number, affinity, or structure of EPOR (28), consistent with posttranslational changes in the abundance or potency of intracellular EPOR peptides that serve to improve signaling efficiency and allow these cell lines to respond to lower levels of EPO. The differential pattern of soluble EPOR peptides seen after PNX may serve a similar purpose.
EPOR and apoptosis.
The apoptosis index in immature dog lung was 4.7-fold that in mature lung and correlates with increased cell proliferative activities previously reported in these same lungs (16). Schittny and colleagues (44) observed increased apoptosis in rat lungs during the third postnatal week, which is thought to promote structural maturation by reducing numbers of fibroblasts and type II cells and thinning the alveolar septa. Our findings likely reflect a similar remodeling process. In these growing animals, the already elevated apoptosis index further increased by
50% 3 wk after PNX above that in age-matched sham controls, associated with further enhanced cellular proliferation (16) and disproportionate expansion of the interstitial compartment (18). Indexes of proliferation and apoptosis subsided by the time animals reached somatic maturity 10 mo post-PNX.
EPO suppresses apoptosis in erythroid precursor cells (28) and increases expression of antiapoptotic proteins Bcl-2 and Bcl-XL in mouse erythroid cell lines (17, 48). One may therefore expect EPO signaling via EPOR to correlate inversely with apoptotic index. Instead, we found increased EPOR expression during both developmental and compensatory lung growth in direct relationship to indexes of cell proliferation as well as apoptosis. Combined observations from in vitro and in vivo studies suggest that EPO signaling may moderate lung growth stimulated by other signals to prevent runaway cell proliferation or apoptosis. Alternatively, EPO signaling may exert additional effects unrelated to apoptosis, such as direct stimulation of angiogenesis, discussed below.
EPOR and angiogenesis.
Hypoxia is a primitive and global stimulus for the growth of gas exchange organs in lower vertebrates (6), rodents (7, 10), dogs (25), and likely humans (12, 13). Hypoxia concurrently stimulates production of circulating EPO and VEGF (14) via transcriptional activation by oxygen-sensitive hypoxia-inducible factor 1 (47). EPO and VEGF are coexpressed during tumor angiogenesis (30), and EPO can substitute for VEGF in stimulating capillary outgrowth from human adult myocardial tissue (23). Thus upregulation of EPOR in lung during development and post-PNX may enhance alveolar capillary growth directly or via interaction with VEGF and/or other angiogenic growth factors. Because alveolar hypoxia is not a feature of postnatal lung maturation and is only mild to moderate in post-PNX animals at rest, there must be an additional signal(s) that can cause EPOR upregulation in lung, for example, mechanical strain imposed on the alveolar septa by a growing thorax during development or by compensatory expansion of the remaining lung after PNX.
In conclusion, we find elevated expression of membrane-bound EPOR in the lung during postnatal maturation and further increased expression during early post-PNX lung growth, whereas various soluble EPOR peptides show different patterns of expression depending on the type of lung growth. These results support the concept of paracrine regulation of alveolar growth via EPO signaling; this novel pathway may potentially integrate developmental, post-PNX, and hypoxia-enhanced alveolar growth. The action of EPOR signaling likely involves limiting excessive cell proliferation and apoptosis induced by other growth signals and/or direct stimulation of alveolar angiogenesis.
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GRANTS
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This work was supported by National Institutes of Health Grants RO1-HL-40070, HL-54060, HL-45716, HL-62873, RO1-DK-48482, and DK-64396 as well as the Department of Veterans Affairs Research Service.
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ACKNOWLEDGMENTS
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The authors thank Dr. Aaron S. Estrera for surgical expertise; Heather L. Stanley, Richard T. Hogg, Debbie C. Tuttle, and Jue Yang for animal care and technical aid; the staff of the Animal Resources Center for veterinary assistance; and Dr. Steven Kernie, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, for assistance with TUNEL assay.
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FOOTNOTES
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Address for reprint requests and other correspondence: C. C. W. Hsia, Dept. of Internal Medicine, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9034
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