ARTICLE |
Correspondence to: Maria I. Ramirez, Boston U. School of Medicine, Evans Biomedical Research Center, X-440, 650 Albany St., Boston, MA 02118. E-mail: mramirez@lung.bumc.bu.edu
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Summary |
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Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5' variant that will translate mainly into caveolin-1 and ß isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1
localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1
is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1
is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1
is also expressed in alveolar Type I cells. (J Histochem Cytochem 50:3342, 2002)
Key Words: lung, caveolin-1, caveolae, blood vessels, endothelium, vasculogenesis, development, alveolar epithelial type I cells
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Introduction |
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Caveolins are a family of integral membrane proteins (caveolin-1, -2, and -3) that are key components of caveolae (
Although caveolae are present in most tissues, caveolin family members are differentially expressed in various cell types. Caveolin-1 mRNA is highly abundant in adult adipose tissue and lung, and is detected at lower levels in other tissues ( and ß) (
-isoform 24 kD and ß-isoform 21 kD) are produced by different translation initiation sites, 32 aa apart (
and ß), caveolin-1 proteins are detected in the adult lung, mainly in alveolar epithelial Type I cells and alveolar capillary endothelial cells (
isoform is expressed in the vasculature but not in the epithelium. In adult lung, expression is detected in endothelial blood vessel cells and alveolar epithelial Type I cells.
The process of vasculogenesis in the lung begins when the lung buds from the foregut on embryonic day 99.5 (E99.5) (
In contrast, there is considerable information about molecules that influence lung epithelial development, including, e.g., many known growth factors and signaling molecules ( (
Previous studies of caveolin expression during mouse development were performed by Northern and Western blotting analysis using RNA or protein extracts from whole embryos starting at day E7. Although these data show that caveolin-1 is expressed very early in mouse embryos, they are uninformative about expression sites in different organs and/or cell types (
The current knowledge about transcriptional and/or post-transcriptional regulation of caveolin-1 expression is limited. Promoter studies show that two sterol regulatory elements mediate the upregulation of caveolin-1 gene transcription in response to increased levels of intracellular free cholesterol (- and ß-isoforms of caveolin-1. In vitro transcription/translation studies showed that full-length caveolin-1 mRNA lacking the 5'UTR could produce either
- or ß-isoforms but that a short sequence of the 5'UTR (22 bp) predominantly drives translation of the
-isoform. The 5' variant mRNA, on the other hand, only produces the ß-isoform (
- and ß-isoforms can form caveolae of different shapes, but it is not clear if the shape of the caveolae could be linked to a difference in function (
We have studied caveolin-1 expression patterns to determine how and when caveolin-1 is targeted to lung cells, particularly alveolar epithelial Type I cells. Our previous work about the regulation of T1 (
is expressed early during lung development in the epithelium and is restricted to Type I cells in the adult lung. It is therefore an excellent molecular tool to follow the Type I cell phenotype in vitro and in vivo. However, to identify common regulatory events that direct the Type I cell phenotype overall, we need additional cell-specific markers.
We show that during lung development two mRNAs able to produce caveolin-1- or ß-isoforms are expressed in the lung. Caveolin-1
protein is expressed in the developing vasculature but not in the epithelium. In the adult, expression is detected in endothelial cells and alveolar epithelial Type I cells. Endothelial expression is detected on E10; epithelial expression first appears postnatally. It is likely, therefore, that the regulatory mechanisms of caveolin-1 gene transcription and/or translation are markedly different in endothelial and epithelial cells of the lung.
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Materials and Methods |
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RNA and Protein Purification
Lungs were isolated from E12, E15, E19, postnatal Day 3, 5, 30, and 60, and adult FVB mice (Charles River; Wilmington, MA) (n=13). They were housed in VFB (virus-free barrier) rooms, using methods described earlier (
RT-PCR
Total RNA (1 µg) was reverse-transcribed using AMV-reverse transcriptase (Promega; Madison, WI) following the manufacturer's protocol in a final volume of 25 µl. Oligonucleotides to amplify the 5' end of the full-length and 5' variant caveolin-1 mRNAs were ex2/R common reverse primer and a-start/F or 5' V/F1 as forward primers described previously by others (
Western Blots
Protein extracts from E15, E19, and adult lung (10 µg) (n=2) were electrophoresed on a 10% polyacrylamide gel by standard methods (
Immunostaining Analysis
Mouse embryos and fetal and adult lungs (n=13) were fixed in 4% paraformaldehyde by immersion for 18 hr and embedded in paraffin by standard methods ( (N-20) antibody in the presence of a five- or 10-fold excess of the blocking peptide (aa 221) (Santa Cruz Biotechnology). Immunostaining was performed with a Vectastain Elite ABC kit (Vector Laboratories) followed by biotinyl/tyramide signal amplification (TSA-Indirect; NEN Life Science Products, Boston, MA) and visualized using DAB (diaminobenzidine) as a substrate. PECAM-1 staining was done using the same method as caveolin-1. An anti-PECAM-1 (M-20) antibody (Santa Cruz Biotechnology) was used at a 1:1000 dilution. Counterstaining was performed using hematoxylin and eosin as previously reported (
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Results |
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Expression of Caveolin-1 mRNAs During Mouse Lung Development
Two messages for caveolin-1 that can be translated mainly into the - or ß-isoforms have been recently described (
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Western Blotting Analysis of Caveolin-1 and PECAM-1 Protein Expression During Mouse Lung Development
Caveolin-1 protein has a unique N-terminal sequence against which the anti caveolin-1 (N-20) antibody was raised (aa 221). Western blots using this antibody (Fig 1B) show only one band in the lung at every developmental time studied; we conclude that this band is caveolin-1
(24 kD). We can detect increasing levels of caveolin-1
protein in E15, E19, and adult lung extracts. This increasing pattern of expression matches that of PECAM-1 (Fig 1B). These Western blots indicate that the antibodies used in this study are specific for the proteins of interest.
Immunohistochemical Analysis of Caveolin-1 Protein Expression in Embryonic and Adult Lung
Using the N-20 (sc-894) antibody, we determined the expression pattern of caveolin-1 protein in embryonic, fetal, postnatal, and adult lung tissue sections. Analyses were performed in sections from one to three embryos or adult lung.
Whole Embryo at E15.
To obtain an overview of caveolin-1 expression in mouse fetus, we examined staining patterns of sagittal sections of an E15 mouse embryo (Fig 2). At this developmental stage, caveolin-1
is mainly, if not entirely, expressed in endothelial cells and the staining patterns clearly outline the vasculature in many organs. Along with the kidney (Fig 2A) and gut (Fig 2A and Fig 2F), the lung appears to express the highest levels of caveolin-1
(Fig 2A, Fig 4D, and Fig 4E). Endothelial cells of the heart are also positive (Fig 2G). There is no detectable expression in liver sinusoids (Fig 2E), although caveolin-1
was shown to be expressed in adult rat liver endothelium by an electron immunogold method (
(Fig 2B and Fig 2C). The choroid plexus, rich in capillaries, does not show any stained cells (Fig 2D) at this developmental time point. Blood vessels in the forming meninges also stained positive for caveolin-1
(Fig 2A).
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Lung Tissues
Embryonic Day 10.
Immunohistochemical studies of the lung at this developmental stage were performed on serial sections. Caveolin-1 is expressed by clustered cells scattered throughout the lung mesenchyme (Fig 3A). Careful examination of the stained areas shows that each cluster appears to be a small vessel with a discernible lumen (Fig 3D and Fig 3E). Staining of serial sections using anti-PECAM-1, a marker for endothelial differentiation (
is indeed expressed in forming blood vessels (Fig 3C). No specific staining is observed in the epithelial bud (Fig 3A and Fig 3B). In competition assays, incubation with anti-caveolin-1 antibody in the presence of a 10-fold excess of the blocking peptide (aa 221) shows that staining of the forming vessels is specific (Fig 3B). Binding of the antibody to the forming vessels can be prevented by incubation with the soluble specific peptide, but staining of the pleura and some regions of the apical membrane of the pulmonary epithelium cannot be blocked, confirming that epithelial and pleural staining is nonspecific. The nonspecific nature of the pleural and epithelial staining is also confirmed by staining with secondary antibody alone (Fig 3F and Fig 3G). The nonspecific staining of some areas of the epithelium and pleura was also observed at other developmental time points (Fig 4F and Fig 4G, Day E15). PECAM-1 has been shown to be expressed in lung endothelial cells starting at E11 (
Embryonic Day 12.
By Day E12 (Fig 4A4C), the expression of caveolin-1 is highly localized to blood vessels and is not detected in epithelium. Vessels of all sizes are positive, including capillaries of the presumptive peripheral lung and large vessels. There is often a well-defined ring of small vessels circumferentially arranged around the developing bronchioles (Fig 4B). Other mesenchymal cells, including smooth muscle, are negative.
Embryonic Day 15. Analysis of lung at higher magnification shows that the epithelium and mesenchyme are negative but intense staining is observed in the forming blood vessels, including capillaries (Fig 4D and Fig 4E). Nonspecific apical staining of some areas of the epithelium and the pleura was also observed at this developmental time point (Fig 4F and Fig 4G).
Embryonic Day 19.
Towards the end of gestation (Fig 5A5C) the peripheral capillary bed is intensely reactive for caveolin-1. Although morphogenesis of Type I cells is ongoing and the cells express the Type I cell marker proteins T1
and Aqp-5 by this time (
expression in either bronchial or distal epithelia. Counterstaining with hematoxylin and eosin shows that only endothelial cells express caveolin-1
, whereas muscle cells surrounding either upper airways (Fig 5D) or blood vessels (Fig 5E and Fig 5F) are negative.
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Neonatal and Adult Lung.
Endothelial staining is readily detected on postnatal Days 1, 3, 5, 30, and 60 (not shown) but epithelial staining is not evident, if present. In adult lungs (Fig 6A), however, there is continuous staining of the alveolar walls in a pattern that matches the expression of the type I cell marker T1 (
, confirming the results of
(Fig 6F6H). Control sections using secondary antibody alone show no staining in the distal lung (data not shown).
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Discussion |
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We present here studies of the developmental pattern of expression of the caveolin-1 gene in normal mouse lung, from early development until adulthood, focusing in the temporal patterns of expression of caveolin-1 protein in endothelium and epithelium. Most of the studies about caveolin-1 published to date describe the functions and distribution of this gene in adult tissues or in cell lines. There is minimal information about expression of caveolin-1 during mouse development, and the patterns of expression of the two isoforms of caveolin-1 protein during development have not been previously reported.
The lung is one of the organs that expresses the highest levels of caveolin-1 mRNA in rodents (- and ß-isoforms of caveolin-1. Our findings show that two caveolin-1 messages, which can be translated into caveolin-1
and -ß proteins, are present in the lung throughout development and that the
-isoform of caveolin-1 is expressed as soon as the lung buds only in endothelial precursors that form the initial network of pulmonary blood vessels.
Considering that the lung starts budding from the foregut endoderm at day E9.09.5, we show expression of endogenous endothelial markers (PECAM-1 and caveolin-1) at the earliest developmental time point of the forming lung studied to date. Another early marker of lung vasculogenesis may be flk-1. Although the endogenous gene pattern in the budding lung was not studied (
Caveolin-1 may therefore be involved in lung endothelial cell differentiation and/or function during vasculogenesis. Early expression of caveolin-1
in the vascular plexus suggests that the caveolin-1 gene could be upregulated by factors that trigger vasculogenesis. Vascular endothelial growth factor (VEGF) and its receptors (Flk-1, Flt-1, and Flt-4) have critical roles in vasculogenesis: Mice carrying null mutations for either Flk-1 receptor or VEGF are deficient in blood vessels and in the specification of endothelial precursors (
is expressed in endothelial cells early during lung development, whether caveolae form in primitive endothelial cells is unknown. The functions of caveolae and caveolin proteins in lung development are also unknown. Downregulation of caveolin-1 expression appears to be necessary for enhanced cell proliferation, as shown by studies of both tumor and oncogene-transformed cell lines (
Caveolin-1 expression remains strictly endothelial in the fetal lung. Postnatally, vascular expression persists with little or no expression in the epithelium. In adult mice (>2 months) expression is clearly observed in vessels and in epithelial Type I cells, as also reported by others. Postnatal upregulation of caveolin-1
in lung epithelium appears to take place gradually. The alveolar spaces of the adult lung are lined by thin, flat epithelial Type I cells that are in close contact with capillaries, providing an extremely large surface for gas exchange (
in Type I epithelial cells and in endothelial cells. Alveolar walls of homozygous caveolin-1 null mice are thicker and hypercellular, and there appears to be hyperproliferation of alveolar endothelial cells. The authors indicate that there is no expression of either caveolin-1
or -ß in null mouse lungs and that caveolae are not formed in alveolar endothelial and Type I epithelial cells. The large number of caveolae and expression of caveolin-1 gene in normal Type I cells and capillary endothelium that form the alveolar wall (
Our studies suggest that - and ß-isoforms of caveolin-1 are expressed in different cell populations during lung development. Caveolin-1
is mostly, if not entirely, expressed in pulmonary endothelial cells before birth, and it does not match the epithelial T1
and AQP-5 gestational expression patterns in the forming lung. Caveolin-1ß could be expressed in the lung epithelium during development because expression of caveolin-1 in the epithelium was described at day E16 using an antibody that recognizes both isoforms. It is possible, therefore, that the caveolin-1ß expression pattern matches those of other Type I epithelial cell genes. The high homology of these two isoforms and the lack of antibodies, produced in species other than mouse, that recognize only the ß-isoform prevent us from characterizing caveolin-1ß expression patterns in mouse lung by immunohistochemistry.
There is an emerging interest in caveolae because of their role in transcytosis, endocytosis, and cell signaling. Interaction of several signaling molecules with the scaffolding domain of caveolin proteins may serve as a mechanism to sequester and inactivate signaling molecules and thus regulate their availability (
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Acknowledgments |
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Supported by NHLBI HL47049 and by the Parker B. Francis Families Foundation. MIR is a Parker B. Francis Fellow in Pulmonary Research.
Received for publication September 22, 2000; accepted August 8, 2001.
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