Developmental regulation of PV-1 in rat lung: association with the nuclear envelope and limited colocalization with Cav-1

Robert Hnasko1 and Nira Ben-Jonathan2

1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; and 2Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati Vontz Center for Molecular Studies, Cincinnati, Ohio

Submitted 25 June 2004 ; accepted in final form 29 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasmalemma vesicle protein-1 (PV-1) is a caveolae-associated protein that is enriched in lung endothelial cells. The PV-1 protein is first detected in the lung at embryonic day 12, before that of caveolin-1 (Cav-1). There is a postnatal rise in PV-1 and Cav-1 mRNA levels, reaching a peak at the time of weaning and declining to their lowest levels in the adult lung. In contrast, the PV-1 protein progressively increases during postnatal development with its highest levels in the adult lung; the Cav-1 protein remains relatively constant throughout this period. Alveolar endothelial cells express both PV-1 and Cav-1 proteins, but PV-1, unlike Cav-1, is also detectable in some bronchial epithelial cells. Endothelial cells transfected with a rat PV-1 construct show a punctate membrane distribution of PV-1, perinuclear accumulation, and an association with the nuclear envelope. In these cells, PV-1 exhibits only partial perinuclear colocalization with Cav-1 and F-actin. In summary, PV-1 is developmentally regulated in the rat lung and shows a divergent intracellular localization, with a limited caveolae/Cav-1 colocalization in cultured endothelial cells.

plasmalemma protein-1; caveolin-1; caveolae; development; endothelial cells; real-time polymerase chain reaction


THE MAJOR PHYSIOLOGICAL FUNCTIONS of the lungs are oxygen uptake and carbon dioxide release. This gas exchange occurs in the alveoli across a single layer of endothelial cells (15). Developmentally, the onset, rate, and duration of alveolar formation require a coordinated and regulated expression of numerous gene products (14, 20, 26, 27). The alveoli are formed in part by septation of saccules that constitute the gas exchange region of the immature lung. In the rat, septation continues until postnatal day 14 and is followed by accelerated thinning of the alveolar wall and conversion of alveolar vessels from a double to a single capillary network (2). The latter process continues at a reduced rate until postnatal day 44 (1).

Disruption of alveolar architecture results in impaired gas exchange and diminished pulmonary function. Clinically, this is observed in premature infants due to insufficient alveolar formation or damage as in pulmonary emphysema (13, 22) and pulmonary fibrosis (28). This underscores the importance of understanding the physiological signals that mediate the normal development of alveolar architecture and its derangement by pathological or environmental factors.

Plasmalemma vesicle protein-1 (PV-1) was originally identified in rat lung membranes as a component of endothelial caveolae (24). It was localized by immunogold electron microscopy to the thin nonmembranous diaphragm that cap some caveolae, suggesting a role for PV-1 in caveolar function (6, 25). Caveolae are plasma membrane organelles that participate in macromolecular transport and intracellular signaling, and they are enriched in lung endothelial cells (19). The caveolin proteins (Cav-1, -2, and -3) are essential structural components of caveolae (7, 17). Cav-1 and Cav-2 are coexpressed in lung endothelial cells and form hetero-oligomeric complexes that interact with a variety of cell signaling molecules (19, 21, 30). Caveolin-deficient mice have vascular abnormalities and restrictive lung disease (9, 17, 18), underscoring the participation of caveolins in a plethora of intracellular processes.

The rat PV-1 gene is composed of 5-exons with one identified mRNA transcript that encodes a 438-amino acid protein with a single span transmembrane domain. PV-1 has a short intracellular NH2-terminal domain and a long N-glycosylated COOH-terminal domain with 10-cysteine residues and two putative coil-coiled domains (24). The mouse and human PV-1 genes share 90 and 70% sequence homology with the rat PV-1 gene, respectively. Neither the PV-1 gene nor protein belongs to any known family (23).

Although PV-1 was initially considered an endothelial cell-specific protein, subsequent studies using antibodies generated in chicken against rat PV-1 have revealed a more diverse cellular distribution, including endocrine cells within the ovary, pancreas, and pituitary (10). Because caveolae are not limited to endothelial cells, PV-1 may be associated with these organelles in numerous cell types. The relative abundance of PV-1 in rat lung alveolar endothelial cells suggests that this protein may serve a role in lung development, maintenance, or function. Therefore, our objectives were to 1) compare the expression of both PV-1 and Cav-1 mRNA and protein in the rat lung during embryonic and postnatal development and 2) examine the intracellular localization of PV-1 in bovine aortic endothelial cells (BAEC) transfected with PV-1 and evaluate its colocalization with endogenous Cav-1.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Timed pregnant Sprague-Dawley rats (Charles River, Wilmington, MA) were housed at the University of Cincinnati, and the procedures involving animals were in compliance with the Institutional Animal Care and Use Committee. The rats were maintained on a 12:12-h light-dark cycle with free access to food and water. Birth was considered postnatal day 1 and weaning occurred at postnatal day 21. Only female rats were used in these studies, and sexual maturation, as defined by vaginal opening, occurred between postnatal days 31 and 34.

PV-1 cloning. The rat PV-1 gene sequence (GenBank accession no. NM_020086) was used to make primers for cloning. Sense primer (nt = 13) 5'-GACACTGCGCAAATGGGGCTC-3' and antisense primer (nt = 1,366) 5'-ACGGGTGGGCGATTCTGGTG-3' were used to amplify full-length PV-1 (1,375 bp) from rat lung by RT-PCR (coding sequence = 25–1,341 nt). Briefly, total RNA from the rat lung was reverse transcribed into cDNA using random hexamers and Superscript II reverse transcriptase (GIBCO-BRL, Gaithersburg, MD). PCR was performed on 1.5 µg of cDNA in the presence of 10% DMSO and 4 mM DTT using Pfu DNA polymerase (Stratagene, La Jolla, CA). The PCR product was verified on a 1% agarose gel by electrophoresis; the band was excised and purified with a QIAquick gel extraction kit (Qiagen, Valencia, CA). EcoRI/Not1 adaptors (Amersham-Pharmacia, Piscataway, NJ) were ligated to the blunt-end PV-1 cDNA, and EcoRI was used for insertion into the pcDNA3.1 vector (Invitrogen, Gaithersburg, MD). Bacterial clones expressing the pcDNA3.1-rPV-1 construct were selected, plasmid was harvested, and DNA sequence analysis was used to verify the orientation and sequence of the rPV-1 insert.

Cell culture and transfection. Primary BAEC were purchased (VEC Technologies, Rensselaer, NY) and grown in EGM-2MV microvascular endothelial cell medium (Cambrex, East Rutherford, NJ) with 5% fetal bovine serum supplemented with endothelial cell attachment factor (Sigma-Aldrich, St. Louis, MO). Cells were transfected with 1 µg of pcDNA3.1-rPV-1 plasmid using FuGENE 6 (Roche, Indianapolis, IN) for 48 h. Cells were either harvested for Western blotting or fixed in 4% paraformaldehyde for subsequent immunofluorescent analysis.

Immunohistochemistry. Sprague-Dawley rat embryos (days 10–18) sectioned and mounted on slides (Novagen, Madison, WI) or rat lungs (postnatal days 1, 10, 22, 35, and 50) were used. Postnatal lungs were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 µm. Immunohistochemistry was performed using affinity-purified chicken anti-rat PV-1 IgY as previously described with some modifications (10). Briefly, deparaffinized sections were subjected to antigen retrieval (29) and blocked with BlokHen (1:5 in PBS; Aves Labs, Tigard, OR) and incubated with primary antibodies: chicken anti-rat-PV-1 (0.56 µg/ml), rabbit anti-Cav-1 (1:2,000; N20 sc-894, Santa Cruz Biotechnology), or rabbit anti-factor VIII-related antigen/von Willebrand factor (vWF; 1:50 following pepsin digestion at 1 mg/ml Tris·HCl, pH 2.0, for 10 min; Zymed, San Francisco, CA) overnight at 4°C. Purified nonimmune IgY (Aves Labs) or chrompure rabbit IgG (Jackson Labs, Bar Harbor, ME) was used as a negative control. Tissue was then incubated for 3 h with biotinylated secondary goat anti-chicken or goat anti-rabbit (0.5 µg/ml; Vector Labs, Burlingame, CA) antibodies followed by incubation for 1 h with avidin-peroxidase (ABC elite, Vector) and signal resolved using a 3,3'-diaminobenzidine substrate (Vector). Sections were counterstained with hematoxylin, cleared in xylene, and coverslipped. Light microscopic images were obtained by Metamorph (Molecular Devices, Sunnyvale, CA) under a Nikon Microphot FXA microscope with a charge-coupled device camera.

Immunofluorescence. BAEC were grown on glass coverslips coated with poly-D-lysine (0.1 mg/ml in distilled H2O, Sigma) and transfected with pcDNA3.1-rPV-1 plasmid for 48 h. Briefly, cells were fixed in 4% paraformaldehyde for 20 min at 4°C, washed in 10 mM PBS (pH 7.2), blocked with BlokHen (1:5 in PBS, Aves Labs) overnight, and incubated for 3 h with chicken anti-rat-PV-1 (0.56 µg/ml) in combination with the following: 1) rabbit anti-Cav-1 N20 (1:3,000; Santa Cruz Biotechnology), 2) rhodamine-phalloidin (1:100; Molecular Probes, Eugene, OR), or 3) preincubation with the endothelial selective acetylated low-density lipoprotein (LDL) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Biomedical Technologies, Stoughton, MA) in complete media at a concentration of 10 µg/ml for 4 h at 37°C before fixation. Nonimmune IgY (Aves Labs) and IgG (Jackson Labs) antibodies were used as negative controls. Immunofluorescent secondary antibodies (1:3,000; Jackson Labs) used include donkey anti-chicken IgG conjugated to fluorescein (FITC) and donkey anti-rabbit IgG. Nuclei were labeled with 1 µg/ml of 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Images were obtained as above with use of appropriate fluorescent filters.

Primers and probes. All primer sets used for real-time PCR span at least one intron-exon boundary: PV-1 sense primer (5'-CGACCTGATCACCTACATAA-3'), antisense primer (5'-CCAGCAGGCTGTCCTTGTCT-3'), and 5'-tetrachlorofluorescein (TET)-PV-1 probe (5'-TET-AAGACCTGCGAAGCCTTGCT-BHQ1–3'). The PV-1 primers correspond to nt 384–583 of rat PV-1 (GenBank accession no. NM_020086). The PV-1 probe (475–494 nt) contains TET fluorophore (539 nm emission maximum) and a 3' Dark Hole Quencher 1 (BHQ1, 538 nm absorption maximum). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as normalization control: sense primer (5'-AAGCTGGTCATCAATGGCAAAC-3'), antisense primer (5'-GAAGACGCCAGTAGACTCCACG-3'), and 5'6-carboxyfluorescein (FAM)-GAPDH probe (5'-FAM-ATCTTCCAGGAGCGAGATCCCG-BHQ1–3'). The GAPDH primers correspond to 1,039–1,149 nt of rat GAPDH (GenBank accession no. AF106860). The GAPDH probe (1,069–1,090 nt) contains FAM fluorophore (520 nm emission maximum) and a 3' BHQ1 quencher. Cav-1 sense primer (5'-GGGTAAATACGTAGACTCCGA-3') and antisense primer (5'-TTGTTGGGCTTGTAGATGTTG-3') correspond to 37–114 nt of rat Cav-1 (GenBank accession no. Z46614). All primers and probes were from Integrated DNA Technologies (Coralville, IA).

Kinetic PCR. Lung tissue, excised at different postnatal developmental stages, was homogenized in TriReagent (Molecular Research, Cincinnati, OH), and RNA was extracted according to the manufacturer's protocol and reverse transcribed using random hexamers and SuperScript II (GIBCO-BRL). The cDNA was subjected to PCR using Taq DNA polymerase (GIBCO-BRL) at a final concentration of 2 mM MgCl2, 10% DMSO, 4 mM DTT, 60 pmol primers with 10 pmol of probes (PV-1 and GAPDH) or with 0.03x of SyberGreen (Molecular Probes). PCR reactions were carried out on a Cepheid Smart Cycler PCR machine (Cepheid, Sunnyvale, CA) using a two-step PCR protocol; denaturation at 96°C for 15 s followed by annealing and extension at 58°C for 45 s. Cycle threshold (Ct) was determined by the inflection point of the reaction growth curve. Quantification was done by the comparative Ct method, and relative gene expression levels were determined with the formula 2{Delta}{Delta}Ct (3, 11, 12). Values are expressed as n-fold relative to rat lung on day 50 (calibrator), which represents the mean of six rat lungs, each run in triplicate. Briefly, the Ct was determined for the target amplicons (PV-1 or Cav-1), and values were normalized against the internal control (GAPDH) to account for differences in total amount of nucleic acid added and the efficiency of the RT step ({Delta}Ct). The {Delta}Ct for each experimental sample was subtracted from the {Delta}Ct of the calibrator ({Delta}{Delta}Ct), and the amount of target, normalized to an internal control and relative to the calibrator, was calculated by 2{Delta}{Delta}Ct and expressed as n-fold difference relative to the calibrator.

Western blotting. Lung tissue was homogenized on ice in an extraction buffer (25 mM HEPES, 150 mM NaCl, and 1% Triton X-100; pH 7.5) containing a protease inhibitory cocktail (Sigma). BAEC were extracted by sonication using the same buffer. Tissue or cell lysates were passed through a 0.75-µm nylon mesh, and the supernatant was centrifuged at 500 rpm for 10 min at 4°C. The cleared lysate was subjected to ultracentrifugation at 65,000 rpm for 2 h at 4°C in a Ti70 rotor, and the pellets were solubilized by sonication in extraction buffer containing 60 mM NaOH. Protein concentration was determined by BCA assay, and the samples were reduced in DTT with 10% 2-mercaptoethanol and boiled in SDS-containing sample buffer. Equal concentration of proteins (50 µg) were loaded on a discontinuous (4–12%) polyacrylamide gel and subjected to electrophoresis. Proteins were transferred to nitrocellulose and blotted for PV-1, Cav-1, or {beta}-actin. Briefly, all Western buffers included 10 mM Tris·HCl (pH 8) containing 0.1% Tween 20; blocking was done with 10% nonfat dry milk. Affinity-purified chicken anti-rat PV-1 antibody was used as previously described (10) at a concentration of 0.56 µg/ml IgY. A polyclonal rabbit anti-caveolin-1 (N20, Santa Cruz Biotechnology) was used at a concentration of 0.08 µg/ml, which detects a single band representing the predominant alpha form of caveolin-1. A monoclonal antibody against {beta}-actin (1:10,000; Sigma clone AC15) was used as an internal loading control. Horseradish peroxidase-conjugated goat anti-chicken (1:30,000; Sigma), goat anti-rabbit (1:20,000; Vector), or goat anti-mouse (1:40,000; Amersham) secondary antibodies were used to detect PV-1, Cav-1, and {beta}-actin proteins, respectively. Enhanced chemiluminescence (ECL, Pierce) was used to resolve proteins on X-ray film (Fuji, SB5). BenchMark prestained protein ladder (Invitrogen) was used to estimate molecular weight.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Detection of PV-1 in the rudimentary alveolar vasculature of the rat lung on embryonic day 12. Figure 1 compares the immunohistochemical localization of PV-1 (left panels) and Cav-1 (right panels) in the rat lung at embryonic days 12, 14, and 18 and birth. PV-1 expression is seen already by embryonic day 12 in endothelial cells lining the primitive lung vasculature (top left panel), whereas Cav-1 is undetectable at this stage of development (top right panel) despite the morphologically evident vascular endothelial cells. Cav-1 expression is seen by embryonic day 14, and like PV-1, its expression is confined to cells lining blood vessels of the embryonic lung. No staining was observed in IgY or IgG control slides (data not shown).



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Fig. 1. Detection of plasmalemma vesicle protein (PV)-1 and caveolin (Cav)-1 protein in rat lung vascular endothelial cells during embryonic development. The PV-1 protein (left panels) is detectable at embryonic day 12 (E12) and Cav-1 protein at E14 (right panels) in lung vascular endothelial cells. Both PV-1 and Cav-1 are localized to cells lining the vascular spaces of alveoli [brown diaminobenzidine (DAB) staining] throughout embryonic lung development (E14–E22) and at parturition [day (d) 1]. Nuclei are counterstained with hematoxylin. Bars = 50 µm.

 
Localization of PV-1 and CAV-1 proteins within the lung during postnatal development. Figure 2 compares the immunohistochemical localization of PV-1 (left panels), Cav-1 (center panels), and vWF (right panels) in the lung at select days (days 1, 22, 35, 50) during postnatal development. Both PV-1 and Cav-1 proteins are seen in capillary endothelial cells of the alveoli at all time points. Only endothelial cells of large pulmonary blood vessels, but not those in smaller capillaries, stain positive for vWF. The presence of PV-1 in cells lining terminal bronchioles of the rat lung on day 50 is shown in Fig. 3A, whereas Cav-1 is not detectable in any bronchial cells (Fig. 3B). In addition, PV-1 (Fig. 3, C and E), but not Cav-1 (Fig. 3, D and F), is detected in ciliated cells of the pseudostratified bronchial epithelium. No staining was observed in IgY and IgG control slides (data not shown).



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Fig. 2. Cellular localization of PV-1, Cav-1, and von Willebrand factor (vWF) proteins in rat lung during postnatal development. PV-1 (left panels) and Cav-1 (center panels) are expressed throughout postnatal development in capillary endothelial cells lining alveoli (brown DAB staining) as well as in larger blood vessels. The vWF protein (right panels) is expressed primarily in cells lining large blood vessels (brown DAB staining) with a limited expression in capillary endothelial cells. Nuclei are counterstained with hematoxylin. Bars = 50 µm.

 


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Fig. 3. Detection of PV-1, but not Cav-1, protein in bronchial epithelial cells from adult rat lung. PV-1 (A, C, E) is detected (brown DAB staining) in cells lining the alveolar vasculature and in some bronchial epithelial cells (black arrows). Cav-1 (B, D, F) is detected (brown DAB staining) in cells lining the alveolar vasculature but is undetectable in bronchial epithelial cells (red arrows). Nuclei are counterstained with hematoxylin. Bars = 50 µm.

 
Progressive increase in PV-1, but not CAV-1, protein in the lung during postnatal development. As determined by Western blotting, the level of PV-1 protein is very low at birth, showing a progressive increase with age that reaches a peak on day 35 and remains elevated on day 50 (Fig. 4). In contrast, the levels of Cav-1 protein remain largely unchanged, with a small rise seen on day 35. A single band of Cav-1 is shown in this blot and represents the full-length {alpha}-isoform. The {beta}-Cav-1 isoform (a 31-amino acid NH2-terminal truncation of {alpha}-Cav-1) is not detected as the Cav-1 antibody used was generated against the extreme NH2 terminus (N20) of full-length Cav-1. Equal protein load was verified by blotting for {beta}-actin. Cell lysate from BAEC transfected with a rat PV-1 construct served as a positive control.



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Fig. 4. Detection of PV-1 and {alpha}-Cav-1 in postnatal rat lung by Western blot. PV-1 shows a developmental increase in the lung with peak levels detected at d35 and d50. The concentration of {alpha}-Cav-1 protein in the lung is stable throughout postnatal development showing a modest rise at d35 and d50. Protein load was normalized by {beta}-actin. Cell lysate from bovine aortic endothelial cells (BAEC), transfected with rat PV-1 cDNA, was used as a positive control. Approximate molecular masses: PV-1, 58 kDa; {alpha}-Cav-1, 22 kDa; {beta}-actin, 42 kDa.

 
Dissimilar changes in PV-1 and CAV-1 mRNA levels in the lung during postnatal development. As shown in Fig. 5, PV-1 mRNA expression increases threefold from day 1 to 22, followed by a gradual decline to day 50. Unlike PV-1, Cav-1 mRNA levels on postnatal day 1 are fourfold higher than those in the adult lung (Fig. 6D). A comparison of the relative changes in PV-1 and Cav-1 mRNA levels at different postnatal days is shown in Fig. 7. Cav-1 mRNA expression from the early postnatal lung (days 1–22) shows higher levels (four- to sixfold) than that on day 50, whereas the developmental rise in PV-1 mRNA levels is of a smaller magnitude (1.5- to 2-fold). Notably, both PV-1 and Cav-1 mRNA levels in the lung peak at weaning and then decline to their lowest levels in the adult lung.



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Fig. 5. Quantification of PV-1 mRNA from rat lung at select time points during postnatal development by real-time PCR. A representative kinetic PCR profile (A) following amplification of PV-1 (solid line) and GAPDH (dashed line) from lung cDNA shows the exponential rise in fluorescence associated with increasing cycle number (dotted line = cycle threshold). Establishment of a linear relationship between increasing concentration of lung cDNA template and decreasing cycle number (B) for PV-1 and GAPDH (points represent cycle threshold) is shown. A standard curve (C) is generated using rat PV-1 cDNA that defines the effective linear range for PV-1 mRNA determination. Quantification of PV-1 mRNA from postnatal lung (D) shows the n-fold change in transcript level relative to d50 adult lung (means ± SE, n = 6).

 


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Fig. 6. Quantification of Cav-1 mRNA from rat lung at select time points during postnatal development by real-time PCR. A representative melting curve (A) for Cav-1 (solid line) and GAPDH (dashed line) shows the presence of a single PCR product (melt peak = plot of first negative derivative of fluorescence vs. temperature) for each optimized primer set using SYBG green dye. A representative kinetic PCR profile (B) following amplification of Cav-1 and GAPDH from lung cDNA shows the exponential rise in fluorescence with increasing cycle number (dotted line = cycle threshold). Establishment of a linear relationship between increasing concentration of lung cDNA template and decreasing cycle number (C) for Cav-1 and GAPDH (points represent cycle threshold) is shown. Quantification of Cav-1 mRNA from postnatal lung (D) shows the n-fold change in transcript level relative to d50 adult lung (means ± SE, n = 6).

 


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Fig. 7. A comparison of PV-1 and Cav-1 mRNA levels in the lung during postnatal development shows changes in transcript magnitude relative to adult levels. Data were replotted from Figs. 5D and 6D.

 
Intracellular localization of PV-1 and CAV-1 in BAEC. As illustrated in Fig. 8, both transfected PV-1 (Fig. 8A) and endogenous Cav-1 (Fig. 8B) show perinuclear colocalization in BAEC (Fig. 8C) but exhibit divergent and punctate localization along the cell periphery and at the membrane surface. F-actin, which is primarily localized to fibers along the cell length (Fig. 8E), shows only limited colocalization with PV-1 in the perinuclear region (Fig. 8F). In BAEC, internalized acetylated LDL showed a typical punctate cytoplasmic accumulation (Fig. 8H), but no colocalization with PV-1 (Fig. 8I). No immunofluorescent signal was detected from cells in IgY or IgG control slides (data not shown).



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Fig. 8. Intracellular localization of rat PV-1 protein expressed in BAEC. PV-1 (FITC, A) and endogenous Cav-1 [tetramethylrhodamine isothiocyanate (TRITC), B] show some perinuclear colocalization (arrow, C). PV-1 (FITC, D) and F-actin (TRITC, E) show limited perinuclear colocalization (F, arrow). PV-1 (FITC, G) and acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (red, H) show no colocalization (I). DAPI (blue) = nucleus. Bars = 50 µm.

 
Association of PV-1 with the nuclear envelope. Figure 9A is an enlarged image of Fig. 8D without DAPI nuclear staining. It shows an asymmetrical perinuclear aggregation of PV-1 and a thin punctate distribution that encircles the nuclear perimeter. In a dividing cell (Fig. 9B), PV-1 is seen at the border between the two nuclei and also outlines the perimeter of each nucleus. A thin rim of PV-1 is seen along the edge of two lobular nuclei (Fig. 9C), with PV-1 localized to a cleft or furrow in one of the nuclei. Note the asymmetrical perinuclear aggregation of PV-1 that appears to form a structure that is tethered to both nuclei. Nuclear staining of PV-1 was not observed in the absence of detergent-mediated membrane permeation (data not shown).



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Fig. 9. Association of PV-1 with the nuclear envelope in BAEC. PV-1 is localized around the nuclear perimeter (FITC, arrows, A) in a discontinuous punctate pattern. PV-1 is localized at the plane of separation between two postmitotic nuclei (yellow arrow, B), along the nuclear perimeter (white arrows), and as aggregates in the cytosol. PV-1 is localized along the entire perimeter of cell nuclei (white arrows, C), in a nuclear furrow (red arrow), and in a perinuclear aggregation associated with the lobular end of two nuclei. DAPI (blue) = nucleus. Bars = 50 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
These data show that PV-1 expression in the rat lung is not restricted to either endothelial cells or caveolae and is developmentally regulated. PV-1 is detectable in the immature lung vasculature as early as embryonic day 12, before the appearance of Cav-1 on embryonic day 14. Both PV-1 and Cav-1 mRNA levels are elevated in the immature postnatal lung compared with adult tissue, with a significant rise seen from birth to day 22 followed by a linear decline to adult levels. In contrast, the PV-1 protein progressively increases during postnatal lung development, reaching its highest levels during adulthood. Cav-1 mRNA levels are relatively high and stable during postnatal lung development, showing a modest rise at day 22 followed by a decline to adult levels at day 50. The Cav-1 protein remains stable during lung development with a slight increase seen following sexual maturation beginning at day 35. In the adult lung, both PV-1 and Cav-1 are detected in alveolar endothelial cells, yet PV-1, but not Cav-1, is also seen in some bronchial epithelial cells. This divergent cellular distribution reveals that PV-1 is not restricted to endothelial cells, is not strictly colocalized with Cav-1, and is not limited to caveolae. Indeed, in vitro expression of PV-1 in endothelial cells confirms its divergent intracellular localization with a limited perinuclear overlap with Cav-1. Moreover, PV-1 shows variable intracellular distribution: 1) punctate, 2) perinuclear, and 3) association with the perimeter of the nuclear compartment.

The embryonic rat lung progresses through three developmental phases, beginning around embryonic day 16 with a glandular stage during which rudimentary lung buds are formed. This is followed by the canalicular stage beginning around embryonic day 18 with the first recognizable acinar region, increased vascularization, and formation of rudimentary air sacs. The final saccular stage begins around embryonic day 20 and continues through the remainder of gestation and is characterized by the appearance of surfactant and further differentiation of the respiratory tree until postnatal day 4 (5). The process of alveolar septation occurs from postnatal day 4–14, and during this time alveoli increase dramatically in number. Throughout postnatal development there is a progressive cytoplasmic thinning of type I pneumocytes and a transition from a double to a single endothelial cell layer that ultimately composes 10 and 40% of the total alveolar cells in adult lung, respectively (1, 2).

The PV-1 protein is detectable in cells lining rudimentary blood vessels at embryonic day 12, before that of Cav-1. This expression pattern suggests PV-1 may play an early role in the differentiation of endothelial cells from angioblasts during vasculogenesis. Earlier time points were not evaluated so exactly when the PV-1 protein is first expressed in the embryo remains undetermined. The Cav-1 protein was detected by embryonic day 16, and like PV-1 it is expressed in endothelial cells through parturition. Both PV-1 and Cav-1 proteins are localized to the capillary endothelial cells of alveoli during postnatal development.

The multimeric vWF is a plasma glycoprotein synthesized by endothelial cells that is commonly used to identify vascular elements within tissues (16). Like PV-1 and Cav-1, the vWF protein is detectable in endothelial cells that line larger blood vessels throughout postnatal lung development. However, vWF does not label a significant number of capillary endothelial cells that line lung alveoli. The vWF participates in the blood clotting cascade through its interaction with platelets, and its negligible expression in capillary endothelial cells underscores a functional difference between endothelial cell populations in small versus larger blood vessels.

PV-1 and Cav-1 mRNA levels vary during postnatal lung development with peak levels detected on day 22 and lowest levels seen in the adult. The transient transcriptional upregulation of the PV-1 and Cav-1 genes at the time of weaning suggests involvement of a positive regulatory signal during this transition. This may reflect the dietary changes associated with the transition from suckling to weaning, which has been described for other genes in the lung (4). However, the subsequent decline in PV-1 and Cav-1 mRNA levels may reflect changes in alveolar cell number or cell ratios before maturation. Alternatively, changes in the stability of the mRNA transcripts and subsequent turnover rate may explain differences in the mRNA levels observed throughout lung development.

The postnatal rise and fall of PV-1 mRNA levels contrast with the progressive accumulation of the PV-1 protein during postnatal lung development. This inverse relationship may reflect both a developmental decline in cell number and a growing alveolar surface area associated with the increased plasma membrane of endothelial cells and type I pneumocytes. The continued vascular differentiation results in both morphological and biochemical changes in endothelial cells that may account for the differences in PV-1 expression during development. Furthermore, elevated adult levels of PV-1 may reflect a developmental accumulation as a result of limited protein turnover. Indeed, PV-1 forms dimers/oligomers through disulfide bonding that may stabilize the protein or limit its proteolytic degradation, thereby resulting in its intracellular accumulation. Similar to PV-1, the Cav-1 mRNA shows a developmental decline, but unlike PV-1, the Cav-1 protein level remains relatively stable. These changes likely reflect the developmental differentiation of the alveolar vasculature as it progresses to a mature lung phenotype. Moreover, the Cav-1 protein does not accumulate, suggesting a proteolytic turnover that results in steady-state levels of Cav-1 in the mature lung. These data reflect that of the predominant full-length Cav-1 {alpha}-isoform with changes in the less abundant truncated {beta}-Cav-1 undetermined.

In the lung, the majority of PV-1 protein is detectable in the alveolar endothelial cells. Although the immunohistochemical technique used in these experiments prohibits definitive assessment of PV-1 localization to type I pneumocytes, continuous staining on both sides of the alveolar septae suggests these cells express PV-1. In the mature lung, PV-1 was localized to some epithelial cells of the bronchiole that were devoid of Cav-1 protein. The expression of Cav-1 is essential for the formation of caveolae, and consequently the localization of PV-1 in these cells must be in association with other intracellular structures (17).

The divergent intracellular distribution of the PV-1 and Cav-1 proteins supports the contention that PV-1 is not restricted to caveolae and/or caveolar diaphragms. Indeed, the PV-1 protein is expressed by embryonic lung endothelial cells earlier than Cav-1. Furthermore, in vitro expression of PV-1 in endothelial cells results in only a limited perinuclear colocalization with Cav-1, likely in the Golgi apparatus. The PV-1 exhibits a varied intracellular distribution that includes punctate aggregates on the plasma membrane, accumulation along the cell periphery, perinuclear accretion, and tight association with the perimeter of the nucleus. The localization of PV-1 in such diverse intracellular compartments suggests a targeted intracellular distribution. The PV-1 protein may function as a tethering factor, belonging to a class of large coil-coiled proteins that modulate the specificity or efficiency of initial vesicular attachment (8).

Perhaps the most striking observation is the close association of PV-1 in some cells with the nuclear envelope. Only some cells expressing PV-1 show nuclear association, a phenomenon that might reflect a regulated signal targeting PV-1 to the nuclear envelope in response to signals initiated by cell division or apoptosis. Additional studies should better define the association of PV-1 with nuclear envelope components, such as the structural lamins or the nuclear pore complex, a structure that resembles fenestrae. Furthermore, it should be determined what cellular signals direct PV-1 to the nuclear compartment.

Future studies will need to address the contribution of the PV-1 protein to endothelial cell differentiation and function. The utilization of the Cav-1-deficient mouse, which has impaired pulmonary function as a result of disrupted cellular architecture (17, 18), should provide an interesting model to evaluate the contribution of PV-1 to cellular function in the absence of caveolae. Additionally, it will be of interest to evaluate PV-1 as a putative tethering factor that may modulate the targeting of vesicles to select intracellular compartments. Indeed, the identification of PV-1 as a component of the plasma membrane, Golgi, and nuclear compartment suggests a dynamic role for PV-1 in intracellular trafficking within select cell populations.


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This work was supported by National Institutes of Health Grants ES-10154, ES-09555, and CA-80920 to N. Ben-Jonathan and ES-07250 as a National Research Service Award to R. Hnasko.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Hnasko, Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: rhnasko{at}aecom.yu.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.


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