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Receptor-Type Protein-Tyrosine Phosphatase μ Is Expressed in Specific Vascular Endothelial Bedsin Vivo
Experimental Cell Research 248, 329 –338 (1999) Article ID excr.1999.4428, available online at http://www.idealibrary.com on
RAPID COMMUNICATION Receptor-Type Protein-Tyrosine Phosphatase m Is Expressed in Specific Vascular Endothelial Beds in Vivo Cesario Bianchi,* ,† ,1 Frank W. Sellke,‡ Robert L. Del Vecchio,§ Nicholas K. Tonks,§ and Benjamin G. Neel* ,‡ *Cancer Biology Program and †Department of Medicine and ‡Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts, 02215; and §Cold Spring Harbor Laboratory, Demerec Building, 1 Bungtown Road, Cold Spring Harbor, New York, 11724-2208
We investigated the localization of receptor-type protein-tyrosine phosphatase m (RPTPm) in tissues by immunofluorescence. RPTPm immunoreactivity was found almost exclusively within vascular endothelial cells. RPTPm was more abundant in the arterial tree than in the venous circulation. This pattern of expression was opposite to that of the von Willebrand factor and demonstrated a lack of difference in expression of VE-cadherin. RPTPm was undetectable in the endocardium. In agreement with previous work on nonendothelial cell lines, RPTPm was exclusively at the lateral aspects of endothelial cells in vivo and at cell– cell contacts as well as ex vivo in two- or three-dimensional endothelial cell cultures, and expression levels were upregulated by cell density. RPTPm was detected in few other cells: bronchial and biliary epithelia and cardiocytes (intercalated discs). Our results identify RPTPm as a new marker of endothelial cell heterogeneity and suggest a possible role in endothelial-specific functions, involving cell– cell contact. © 1999 Academic Press
Key Words: signal transduction; angiogenesis; tyrosyl phosphorylation; contact inhibition; cell junctions.
INTRODUCTION
Protein tyrosyl phosphorylation plays an important role in cell proliferation, differentiation, and migration. Tyrosyl phosphorylation is coordinately regulated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). Several endothelial cell-specific PTKs have been identified. For example, TEK is a receptor tyrosine kinase (RTK) expressed in endothelial progenitor cells and in the endothelium of actively 1
To whom correspondence and reprint requests should be addressed at 330 Brookline Ave., DA-801, Boston, MA 02215. Fax: (617) 667-4898. E-mail: [email protected].
growing blood vessels [1–3]. Abrogation of TEK function by gene disruption or by the use of dominant negative mutants causes embryonic lethality, with profound defects in the vasculature [4]. KDR, the human receptor for VEGF (flk-1 in the mouse), is predominantly expressed in endothelial cells, and gene disruption experiments also have revealed its critical role in endothelial cell physiology [5]. These data suggest an important role for reversible tyrosine phosphorylation in the control of endothelial cells. However, little is known about the function of endothelial cell PTPs. A large number of PTPs have now been identified in mammalian species. As with PTKs, both transmembrane, receptor-like PTPs (RPTPs) and non-transmembrane (nonreceptor) PTPs exist. The ectodomains of many RPTPs display a high degree of sequence similarity to cell adhesion molecules [6]. For example, the ectodomain of RPTPm contains four fibronectin type III repeats, an immunoglobulin domain, and a so-called MAM (Meprin, Xenopus A5, MU) domain. Previous work has shown that the ectodomain of RPTPm can promote cell adhesion [7, 8] and revealed a direct association between RPTPm and several cadherins [9, 10], strongly suggesting that RPTPm may transduce signals generated by cell– cell contact in vivo. Consistent with such a notion, studies of tissue culture cells have shown that PTPm colocalizes with cadherin/catenin complexes at cell– cell junctions, and its expression increases markedly with increased cell contact [11]. Biochemical analyses reveal a direct association between RPTPm and several cadherins [9, 10]. Northern blotting experiments reveal that RPTPm is expressed in most tissues examined, but at particularly high levels in brain, heart, and lung [12]. In situ hybridization studies of rat embryonic brain sections show exclusive localization to capillaries [13]. Very recent work comparing mRNA expression of RPTPm, RPTPk, and PCP2 (a.k.a. PTPp or PTPl) in mouse development and in adult brain confirms the almost
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TABLE 1 Distribution of Immunoreactive RPTPm Tissue/Organ Brain Carotid arteries Aorta Heart Lung Skeletal muscle Stomach, intestines Kidneys Adrenals Spleen Liver Umbilical cord
Cell type Endothelial cells of pia-arachnoid arterioles and capillaries Endothelial cells Endothelial cells Endothelial cells, intercalated discs undetectable in endocardium Endothelial cells, bronchi epithelium Endothelial cells Endothelial cells of small arteries in the submucosa Arterial/arteriolar endothelium, glomeruli/small vessels (weak) Arterial/arteriolar endothelium Hilar/central arterial endothelium Arterial endothelium, biliary duct epithelium Arterial endothelium, weak to undetectable in the vein
exclusive expression of RPTPm transcripts in endothelial cells [14]. However, localization of RPTPm protein in vivo has remained unknown. Since studies of tissue culture cells indicate that RPTPm expression is regulated, at least in part, at the posttranslational level [11], the expression of RPTPm RNA and protein in vivo might differ. In this paper, we survey the in vivo expression of RPTPm in multiple tissues from rats, pigs, and humans by indirect immunofluorescence. Our results demonstrate that RPTPm is expressed almost exclusively at sites of cell– cell contact in the vascular endothelium. Moreover, RPTPm expression within the endothelium is heterogeneous: expression occurs predominantly in large arteries compared to capillaries and veins and is undetectable in the endocardium. This pattern of expression only partly overlaps previously published studies of RPTPm mRNA expression during rodent development, suggesting that posttranslational mechanisms may help regulate RPTPm expression in vivo. Our results identify PTPm as a new marker of endothelial cell heterogeneity and suggest that it plays a role in the regulation of endothelial cell-specific tyrosyl phosphorylation events. MATERIALS AND METHODS Tissue preparation. Wistar rats (;250 g) or Yorkshire swines (;25 kg) were euthanized under anesthesia, and tissues were rapidly dissected, frozen in dry ice, and mounted on specimen holders using tissue-freezing media (Triangle Biomedical Sciences, Durham, NC). Umbilical cords were harvested from cesarian section deliveries at Beth Israel Deaconess Medical Center following an IRB-approved protocol. Seven- to 12-mm frozen sections were obtained by using a cryostat (Jung Frigocut 2800N, Leica Inc., Deerfield, IL) and mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA).
Cell culture. Bovine aortic endothelial cells (BAE) were cultured in DMEM (Mediatech, Hendon, VA) supplemented with 10% bovine calf serum (CS) (Hyclone Laboratories, Logan, UT). Cells were plated at 50% confluence on 1.5% bovine gelatin (Sigma Co., St. Louis, MO)-coated eight-well glass chamber slides (NUNC Inc., Naperville, IL) and allowed to grow to confluency. To promote formation of capillary-like structures, confluent BAE were plated on gelatincoated glass slides and fed every 2 days with fresh media containing 10% CS until spontaneous sprouting was observed (typically 7–10 days). Indirect immunofluorescence. All steps were performed at room temperature (RT). For tissue staining, cryosections were air dried for 30 min and fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.6) (PBS) for 20 min. Excess fixative was washed away with PBS, and the tissues were permeabilized with 1% sodium dodecyl sulfate (SDS) in PBS for 5 min [15], washed twice briefly with PBS, and blocked with 1% bovine serum albumin (BSA) (Fraction V, Sigma Co.), 0.02% saponin (Sigma Co.) (blocking buffer) for 60 min before incubation with primary antibodies. For staining of cultured cells, the growth medium was aspirated and the chambers were washed once with PBS. The cells were then fixed for 20 min in fresh 4% paraformaldehyde in PBS and subsequently washed five times with PBS. This incubation was followed by permeabilization and blocking as described above. Primary antibodies were added to the chambers at appropriate dilutions in blocking buffer [anti-RPTPm antibody SK-15 (1:1000 dilution of ascites)], g-catenin (1:2000), b-catenin (1:3000), anti-vWF (Sigma Co.) (1:500), and anti-VE-cadherin (1:500) (Chemicon International Inc., Temecula, CA) for 1 h, followed by three washes with PBS and incubation with the appropriate secondary antibodies, either goat anti-rabbit IgG coupled to FITC (1:200) or donkey anti-mouse IgG coupled to Cy3 (1:500) (Jackson Immunoresearch Laboratories, West Grove, PA), for 30 min. Excess secondary antibodies were removed by washing five times with PBS, and the slides were mounted in Vectashield media (Vector Laboratories, Burlingame, CA). Images were obtained with an Olympus upright microscope. Photographs were taken with Kodak TMAX 400 ASA film at the same exposure time for all pairs of micrographs and were developed and printed under identical conditions. Immunoadsorption experiments with GST alone or GST–RPTPm [16] were performed by preincubating the primary antibodies (three times for 20 min each time) with 10 –20 mg of GST or GST–RPTPm immobilized on a PVDF membrane (Immobilon-P, Millipore Corp., Bedford, MA) at RT before applying them to the tissue sections. Control experiments with the secondary antibody alone were routinely performed and, unless otherwise stated, produced no significant background (data not shown). Immunoblotting. Total cell extracts were prepared from sparse or confluent BAE as follows: The dishes were washed in cold PBS, scraped with a rubber policemen, pelleted in a table-top centrifuge, and ressuspended in water. Aliquots were taken to measure protein using a BCA protein assay kit (Pierce, Rockford, IL) and the remaining portion was lysed directly in Laemmli buffer (62.5 mM Tris–HCl, 2% SDS, 1% b-mercaptoethanol, 10% glycerol, and 0.0025% bromophenol blue) [17]. The lysate was boiled for 5 min, and ;10 mg / 20 ml was subjected to 8% SDS–polyacrylamide gel electrophoresis (SDS– PAGE). Proteins were transferred to Immobilon-P using a semidry blotting apparatus (Bio-Rad, Richmond, CA), membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.1% Tween 20) for 1 h at RT, and the blots were probed with mouse monoclonal anti-RPTPm antibody BK-2 (1:3000 dilution of ascites) in TBST plus 5% milk overnight at 4°C. Unbound antibodies were removed by three 5-min washes in TBST, followed by incubation for 30 min with horseradish peroxidase-coupled antimouse antibodies (Amersham, Little Chalfont, Buckinghamshire, UK) at 1:3000 dilution. Following washing, bound antibodies were visualized by enhanced chemiluminescence (ECL) using Hyperfilm
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FIG. 1. Localization of RPTPm in the rat vascular endothelium. Frozen sections from rat carotid artery (A) and aortic vasa vasorum (B) were prepared and immunostained with antibody SK-15 as described under Materials and Methods. Specificity of RPTPm staining in rat left ventricle sections is shown by comparison of SK-15 immunostaining following (C) preincubation with GST alone or (D) preincubation with GST–C-terminal RPTPm. Arrows (A, B) show staining at sites of cell– cell junction. Note that staining of endothelial cells (small arrows in C) and intercalated discs (larger arrows in C) is blocked upon preincubation of SK15 with its immunogen (D). L, lumen; M, muscularis; E, endothelium; C, capillary. Bar 5 10 mm.
(Amersham). To verify proper fractionation and transfer of proteins, membranes were stained with 0.25% Coomassie brilliant blue R-250 (Sigma Co.) for 2 min, followed by a 10-min wash with 25% methanol/7% acetic acid solution [18].
RESULTS
Expression of RPTPm in the Vascular Tree Expression of RPTPm in various tissues was assessed by indirect immunofluorescence using a monoclonal antibody (SK-15) directed against its first (i.e., more N-terminal) PTP domain. The generation, preparation, and characterization of this antibody have been described previously [7, 10, 16]. It is monospecific for detecting RPTPm in a variety of endothelial cells of different origin (including different species). Expres-
sion was detected in all tissues sampled, but within most immunoreactive RPTPm was restricted to the vascular endothelium (Table 1; see figures below). In the vasculature, RPTPm immunostaining was prominent in large and small arteries (Fig. 1A), their immediate tributaries to arterioles, and the vasa vasorum (Fig. 1B). SK-15 immunoreactivity was unaffected by preincubation with GST alone (Fig. 1C), but immunoreactivity was completely eliminated by preincubation with a GST–RPTPm fusion protein (Fig. 1D), confirming the specificity of our immunoreagents. Notably, RPTPm expression was undetectable in most capillary beds (e.g., kidney, spleen, liver, adrenal), with the exception of those in heart, brain, and skeletal muscle (Figs. 2A and 2B and data not shown). The expression of RPTPm in these capillary beds may reflect particular features
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herins in lysates from cultured cells and primary tissues. In addition, previous studies have shown that RPTPm expression in tissue culture cells is restricted to cell– cell junctions, where it colocalizes with cadherin– catenin complexes [9, 10]. We noted that RPTPm immunoreactivity in tissue sections was restricted to endothelial cell junctions (Figs. 1–5). Consistent with the previous observations in cultured cells, RPTPm also colocalized with b-catenin, g-catenin/plakoglobin, and cadherins, as revealed by double indirect immunofluorescence studies (Figs. 2A and 2B; see also Figs. 7A and 7B and data not shown). RPTPm Expression outside the Vascular System In addition to cardiac arteries and capillaries (Fig. 1), strong RPTPm immunoreactivity was observed in intercalated discs of the heart (Figs. 1C, 3A, and 6A), which do not stain anti-vWF (Figs. 3B and 6B). Interestingly, colocalization of RPTPm and components of
FIG. 2. RPTPm and g-catenin colocalize in endothelial cells. Double immunofluorescence of RPTPm (A) and g-catenin (B) in the vasculature of rat skeletal muscle. Note the colocalization of staining with both antibodies at areas of cell– cell contact (arrows) in an arteriole. L, lumen. Bar 5 10 mm.
of their junctions (see Discussion). RPTPm expression also was undetectable in the endocardium. In the same sections, endocardial cells were easily visualized by anti-von Willebrand factor (vWF) antibodies in double indirect immunofluorescence experiments (Figs. 3A and 3B). Interestingly, the endocardium and other endothelial cells are ontogenetically distinct (see Discussion). RPTPm expression was undetectable in most veins, although immunoreactivity was observed in some larger veins (Figs. 4A and 4B and data not shown). However, even in these, PTPm immunoreactivity was much less intense than in arteries of similar caliber (Figs. 4A and 4B). These differences between arterial and venous RPTPm and vWF immunoreactivities were recapitulated in human umbilical cords (Figs. 5A–5D) despite the lack of difference of adherens junctions as determined by VE-cadherin staining in consecutive sections of the same cords (Figs. 5E and 5F) (see below). RPTPm co-immunoprecipitates with several cad-
FIG. 3. Lack of detectable expression of RPTPm in the endocardium. Frozen sections of the rat ventricular free wall were immunostained for RPTPm and vWF as described under Materials and Methods. Note the absence of RPTPm staining (A) in the endocardium (arrows), compared with the intense staining for vWF (B). i, intercalated discs; C, capillaries. Bar 5 10 mm.
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FIG. 4. Heterogeneity of RPTPm expression in different vascular beds. Immunostaining for RPTPm (A) and vWF (B) in the vasculature of the rat adrenal capsule and its surrounding fat. Note the dramatic difference in staining intensity between RPTPm (A) in endothelial cells in the arteriolar (a) and venous (v) trees, compared to vWF (B). M, medial muscular layer. Bar 5 100 mm.
cadherin/catenin complexes was also observed in these structures (data not shown). RPTPm was also detected at low levels at sites of cell– cell contact in the bronchial epithelium (Figs. 7A and 7B) and in the biliary tract epithelium (data not shown).
RPTPm Expression in Endothelial Cell Cultures Previous studies have examined endogenous RPTPm protein levels in Mv1 Lu cells [9] and heterologously expressed RPTPm in a fibroblast cell line [11]. Our data
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FIG. 5. Lack of difference in VE-cadherin-containing adherens junctions despite marked differences of RPTPm expression in umbilical cord arterial and venous circulations. Frozen sections of a human umbilical cord were immunostained for RPTPm (A, C) and vWF (B, D) (double labeling) in the umbilical vein (B, D) and artery (A, C) and compared with VE-cadherin staining in umbilical artery (E) and vein (F). Note the dramatic difference in staining intensity between RPTPm in endothelial cells in the artery (C) and vein (D) compared with a consecutive section stained with VE-cadherin [artery (E) and vein (D)]. L, lumen; M, muscular layer; endothelium, arrows.
indicate that RPTPm expression in vivo is largely restricted to endothelial cells. Therefore, we monitored RPTPm expression in primary ex vivo endothelial cell cultures. In subconfluent cultures, little RPTPm could be detected at the cell surface, with weak expression primarily confined to a perinuclear region that most
likely represents the Golgi apparatus (Fig. 8A). Consistent with the in vivo studies, RPTPm expression was found at sites of cell– cell junction in confluent BAE cultures (Fig. 8B). Corroborating these immunofluorescence results, immunoblotting experiments confirmed that total RPTPm protein levels are upregulated by cell
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rens junctions between umbilical vein and umbilical arteries as determined by VE-cadherin staining. DISCUSSION
Endothelial cells perform multiple important functions. They are essential for maintaining normal homeostasis, for responding appropriately to inflammatory stimuli, and for controlling endothelial cell permeability to macromolecules [20 –23]. These processes require that proper endothelial junctions be dynamically maintained. Earlier work has shown that signaling via PTKs such as the VEGF receptor can modify vascular permeability [24]. However, the role of specific PTPs in regulating these processes has remained obscure. In this study, we have identified RPTPm as a PTP expressed almost exclusively at endothelial cell junctions in vivo. We find that RPTPm expression is variable in different endothelial cell beds,
FIG. 6. RPTPm expression in intercalated discs. Frozen sections from the rat left ventricle were prepared and immunostained for RPTPm expression (A) and vWF (B), as described under Materials and Methods. Note the staining of intercalated discs (arrows) by RPTPm antibodies, which is not observed with vWF antibodies. C, capillaries. Bar 5 10 mm.
density (Fig. 8C), establishing a direct relationship between cell density, RPTPm protein levels, and RPTPm surface expression in endothelial cells. Note that in endothelial cells, RPTPm undergoes correct processing, generating the mature proteolytically cleaved form, as well as the immature precursor [19], whereas cleavage does not occur detectably in transiently transfected COS cells. Finally, when BAE cells are left confluent for several days, spontaneous sprouting develops, resulting in the formation of a three-dimensional network of tubular structures. As expected from the above studies, RPTPm staining was located predominantly in the cell– cell contacts of these cord-like structures (Fig. 9), reproducing the localization observed in intact vessels. In umbilical cords this heterogeneity is maintained, i.e., despite the presence of venous blood in the arterial tree and arterial blood in the vein (pO 2 levels). In addition, there was no appreciable difference in adhe-
FIG. 7. RPTPm expression and colocalization with catenins in the bronchial epithelium. Double immunofluorescence showing the localization of RPTPm (A) and b-catenin (B) in the rat bronchial epithelium. Note the presence of RPTPm in the bronchi epithelium (arrows in A) which colocalizes with b-catenin (arrows in B). L, lumen; SM, smooth muscle. Bar 5 10 mm.
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Moreover, we find that the RPTPm protein expression pattern only partly overlaps the pattern of expression of its mRNA, arguing that RPTPm expression is regulated by posttranslational as well as transcriptional mechanisms. Our data suggest that RPTPm may function in transducing signals from cell– cell junctions in endothelial cells. Alternatively, or in addition, RPTPm may help control the integrity of endothelial cell junctions. In this regard, preliminary studies indicate that VEGF stimulation causes redistribution of RPTPm expression (R.D.V. and N.K.T., unpublished observations). RPTPm is expressed in the endothelium of largecaliber arteries and in selected capillary beds, providing yet another example of endothelial cell heterogeneity. Multiple studies have shown that endothelial cells in different anatomical locations express distinct molecular markers [22]. In some cases, this heterogeneity
FIG. 8. Modulation of RPTPm expression and compartmentalization in endothelial cell cultures. Bovine aortic endothelial cells cultured on eight-well glass chamber slides either sparsely (A) or at high density (confluent) (B) were immunostained for RPTPm expression. Note the variation in intensity of RPTPm staining and its localization with changes in cell density. (C) Immunoblot of BAE total cell lysates probed with anti-RPTPm monoclonal antibody BK-2. (Left) Two independent experiments comparing BAE under confluent (c) versus sparse (s) conditions. For quantitation, one-half (c/2) and one-quarter (c/4) of the amount of protein from a confluent culture were also loaded. Note the difference in RPTPm protein levels in sparse (s) versus confluent (c) cells. (Right) Effect of increasing culture time on RPTPm expression. BAE were seeded at approximately 50% confluency, (50%) and allowed to grow for 2, 5, or 10 days, as indicated, before determination of RPTPm expression levels. COS cells transiently transfected with RPTPm serve as positive controls for immunoblotting. The size of RPTPm in transfected COS cells is different because of incomplete maturation. N, nuclei; arrows, cell– cell contacts; c, confluent; s, sparse.
with expression much more prominent in large and small arteries than in veins and capillaries, and that its surface expression is regulated by cell density.
FIG. 9. Localization of RPTPm in BAE undergoing tube formation ex vivo. (A) Localization of RPTPm at sites of cell– cell contacts (arrows) in cord-like structures of BAE induced to undergo tube formation (see Materials and Methods for details). (B) Phase contrast of A. Bar 5 10 mm.
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correlates with morphological differences that, in turn, correlate with differences in vascular permeability. For example, the continuous endothelium lining the central nervous system expresses distinct markers compared with the discontinuous endothelium found at other anatomical sites [22]. RPTPm protein expression is highest in the continuous endothelium present in tissues such as skeletal muscle, heart, and lungs. Conversely, little RPTPm protein is expressed in organs with discontinuous (i.e., fenestrated) endothelium, such as the adrenals, gastrointestinal tract, or renal glomeruli, and RPTPm is undetectable in leaky endothelial cell beds, such as those found in the venous circulation of the liver and spleen. This strong correlation between RPTPm protein expression and the presence of endothelial junctions (i.e., cell– cell contact) is consistent with the observation that RPTPm protein levels and surface expression are highly regulated by cell– cell contact (i.e., cell density) in ex vivo cultured cells (Fig. 8). However, the mere presence of cadherin-containing junctions is not sufficient to account for RPTPm expression. In umbilical cord vessels (arteries and veins), the expression of the adherens junction endothelial cell marker (VE-cadherin) is quite similar, yet differences in RPTPm immunoreactivity were dramatic in the arterial compared to venous endothelium and other vascular territories. It should also be pointed out that in the umbilical cord venous blood circulates through umbilical arteries whereas arterial blood circulates through umbilical veins. Therefore, it seems that differences in blood pH and oxygenation alone can not determine whether or not RPTPm is expressed at the EC surface. A recent study monitored RPTPm mRNA expression by in situ hybridization during early mouse development [14]. RPTPm mRNA expression is weak and diffuse at stages 8.5 and 9.5. During embryonic days 12.5 and 14.5, transcription in developing blood vessels is very prominent; similar results were obtained in rat embryos at embryonic day 13.5 [13]. Expression is also observed at some extravascular sites during development, such as the brain parenchyma, particularly the olfactory bulb and cerebellum. By embryonic day 18.5, RPTPm expression was no longer generalized throughout the vasculature, with expression restricted to the heart, kidneys, brown fat in the neck, and, most intensely, lungs. Our work defining the pattern of RPTPm protein expression in adult rats reveals both similarities and some differences with the in situ hybridization studies. We find that RPTPm is expressed in the vasculature of many organs, but shows a particular preference for the arterial bed and capillary territories of the heart, skeletal muscle, and brain. Moreover, we detect no expression of RPTPm in the brain parenchyma, although we did find expression in two
sites (bronchial epithelium and biliary ducts) not observed in the earlier work. It is not clear whether the differences between RPTPm mRNA expression in E18.5 mice and RPTPm protein expression in adult rats reflect further transcriptional regulation of RPTPm during later mouse development, additional, posttranslational mechanisms of regulating RPTPm expression, and/or species-specific differences. Although RPTPm protein is highly expressed in nearly all endothelial cells that form cell junctions, endocardial cells provide a notable exception. Interestingly, previous studies have shown that endocardial cells have a distinct ontogenetic origin from those of nearly all other endothelial cells, deriving from the more axial mesoderm [22]. Since cell– cell junctions are abundant in the endocardium, the absence of RPTPm expression in these cells presumably reflects transcriptional or posttranscriptional regulation, perhaps due to the distinct developmental origin of endocardial cells. Previous work showed that endogenous RPTPm is expressed at points of cell– cell junction in Mv1 Lu epithelial cells [9]. Since we find that bronchial epithelial cells express RPTPm (Fig. 7A). Mv1 Lu cells, which were established from a mink lung, most likely derive from cells of similar origin [28]. Our data support a role for RPTPm in regulating cell– cell junctions in the bronchial epithelium, most likely through interactions with cadherin/catenin complexes. Given the known function of tyrosyl phosphorylation in the dynamic regulation of cell– cell junctions, RPTPm is likely to play an important role in controlling junctional integrity in response to various stimuli. Future studies will be required to define the precise signaling pathways and targets of RPTPm in endothelial and epithelial cell junctions. This work was supported by P01 HL56993-01 (C.B. and B.G.N.), HL-46716 (F.W.S.), and GM 55989 (N.K.T.).
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Received December 16, 1998 Revised version received February 4, 1999
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RAPID COMMUNICATION Receptor-Type Protein-Tyrosine Phosphatase m Is Expressed in Specific Vascular Endothelial Beds in Vivo Cesario Bianchi,* ,† ,1 Frank W. Sellke,‡ Robert L. Del Vecchio,§ Nicholas K. Tonks,§ and Benjamin G. Neel* ,‡ *Cancer Biology Program and †Department of Medicine and ‡Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts, 02215; and §Cold Spring Harbor Laboratory, Demerec Building, 1 Bungtown Road, Cold Spring Harbor, New York, 11724-2208
We investigated the localization of receptor-type protein-tyrosine phosphatase m (RPTPm) in tissues by immunofluorescence. RPTPm immunoreactivity was found almost exclusively within vascular endothelial cells. RPTPm was more abundant in the arterial tree than in the venous circulation. This pattern of expression was opposite to that of the von Willebrand factor and demonstrated a lack of difference in expression of VE-cadherin. RPTPm was undetectable in the endocardium. In agreement with previous work on nonendothelial cell lines, RPTPm was exclusively at the lateral aspects of endothelial cells in vivo and at cell– cell contacts as well as ex vivo in two- or three-dimensional endothelial cell cultures, and expression levels were upregulated by cell density. RPTPm was detected in few other cells: bronchial and biliary epithelia and cardiocytes (intercalated discs). Our results identify RPTPm as a new marker of endothelial cell heterogeneity and suggest a possible role in endothelial-specific functions, involving cell– cell contact. © 1999 Academic Press
Key Words: signal transduction; angiogenesis; tyrosyl phosphorylation; contact inhibition; cell junctions.
INTRODUCTION
Protein tyrosyl phosphorylation plays an important role in cell proliferation, differentiation, and migration. Tyrosyl phosphorylation is coordinately regulated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). Several endothelial cell-specific PTKs have been identified. For example, TEK is a receptor tyrosine kinase (RTK) expressed in endothelial progenitor cells and in the endothelium of actively 1
To whom correspondence and reprint requests should be addressed at 330 Brookline Ave., DA-801, Boston, MA 02215. Fax: (617) 667-4898. E-mail: [email protected].
growing blood vessels [1–3]. Abrogation of TEK function by gene disruption or by the use of dominant negative mutants causes embryonic lethality, with profound defects in the vasculature [4]. KDR, the human receptor for VEGF (flk-1 in the mouse), is predominantly expressed in endothelial cells, and gene disruption experiments also have revealed its critical role in endothelial cell physiology [5]. These data suggest an important role for reversible tyrosine phosphorylation in the control of endothelial cells. However, little is known about the function of endothelial cell PTPs. A large number of PTPs have now been identified in mammalian species. As with PTKs, both transmembrane, receptor-like PTPs (RPTPs) and non-transmembrane (nonreceptor) PTPs exist. The ectodomains of many RPTPs display a high degree of sequence similarity to cell adhesion molecules [6]. For example, the ectodomain of RPTPm contains four fibronectin type III repeats, an immunoglobulin domain, and a so-called MAM (Meprin, Xenopus A5, MU) domain. Previous work has shown that the ectodomain of RPTPm can promote cell adhesion [7, 8] and revealed a direct association between RPTPm and several cadherins [9, 10], strongly suggesting that RPTPm may transduce signals generated by cell– cell contact in vivo. Consistent with such a notion, studies of tissue culture cells have shown that PTPm colocalizes with cadherin/catenin complexes at cell– cell junctions, and its expression increases markedly with increased cell contact [11]. Biochemical analyses reveal a direct association between RPTPm and several cadherins [9, 10]. Northern blotting experiments reveal that RPTPm is expressed in most tissues examined, but at particularly high levels in brain, heart, and lung [12]. In situ hybridization studies of rat embryonic brain sections show exclusive localization to capillaries [13]. Very recent work comparing mRNA expression of RPTPm, RPTPk, and PCP2 (a.k.a. PTPp or PTPl) in mouse development and in adult brain confirms the almost
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TABLE 1 Distribution of Immunoreactive RPTPm Tissue/Organ Brain Carotid arteries Aorta Heart Lung Skeletal muscle Stomach, intestines Kidneys Adrenals Spleen Liver Umbilical cord
Cell type Endothelial cells of pia-arachnoid arterioles and capillaries Endothelial cells Endothelial cells Endothelial cells, intercalated discs undetectable in endocardium Endothelial cells, bronchi epithelium Endothelial cells Endothelial cells of small arteries in the submucosa Arterial/arteriolar endothelium, glomeruli/small vessels (weak) Arterial/arteriolar endothelium Hilar/central arterial endothelium Arterial endothelium, biliary duct epithelium Arterial endothelium, weak to undetectable in the vein
exclusive expression of RPTPm transcripts in endothelial cells [14]. However, localization of RPTPm protein in vivo has remained unknown. Since studies of tissue culture cells indicate that RPTPm expression is regulated, at least in part, at the posttranslational level [11], the expression of RPTPm RNA and protein in vivo might differ. In this paper, we survey the in vivo expression of RPTPm in multiple tissues from rats, pigs, and humans by indirect immunofluorescence. Our results demonstrate that RPTPm is expressed almost exclusively at sites of cell– cell contact in the vascular endothelium. Moreover, RPTPm expression within the endothelium is heterogeneous: expression occurs predominantly in large arteries compared to capillaries and veins and is undetectable in the endocardium. This pattern of expression only partly overlaps previously published studies of RPTPm mRNA expression during rodent development, suggesting that posttranslational mechanisms may help regulate RPTPm expression in vivo. Our results identify PTPm as a new marker of endothelial cell heterogeneity and suggest that it plays a role in the regulation of endothelial cell-specific tyrosyl phosphorylation events. MATERIALS AND METHODS Tissue preparation. Wistar rats (;250 g) or Yorkshire swines (;25 kg) were euthanized under anesthesia, and tissues were rapidly dissected, frozen in dry ice, and mounted on specimen holders using tissue-freezing media (Triangle Biomedical Sciences, Durham, NC). Umbilical cords were harvested from cesarian section deliveries at Beth Israel Deaconess Medical Center following an IRB-approved protocol. Seven- to 12-mm frozen sections were obtained by using a cryostat (Jung Frigocut 2800N, Leica Inc., Deerfield, IL) and mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA).
Cell culture. Bovine aortic endothelial cells (BAE) were cultured in DMEM (Mediatech, Hendon, VA) supplemented with 10% bovine calf serum (CS) (Hyclone Laboratories, Logan, UT). Cells were plated at 50% confluence on 1.5% bovine gelatin (Sigma Co., St. Louis, MO)-coated eight-well glass chamber slides (NUNC Inc., Naperville, IL) and allowed to grow to confluency. To promote formation of capillary-like structures, confluent BAE were plated on gelatincoated glass slides and fed every 2 days with fresh media containing 10% CS until spontaneous sprouting was observed (typically 7–10 days). Indirect immunofluorescence. All steps were performed at room temperature (RT). For tissue staining, cryosections were air dried for 30 min and fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.6) (PBS) for 20 min. Excess fixative was washed away with PBS, and the tissues were permeabilized with 1% sodium dodecyl sulfate (SDS) in PBS for 5 min [15], washed twice briefly with PBS, and blocked with 1% bovine serum albumin (BSA) (Fraction V, Sigma Co.), 0.02% saponin (Sigma Co.) (blocking buffer) for 60 min before incubation with primary antibodies. For staining of cultured cells, the growth medium was aspirated and the chambers were washed once with PBS. The cells were then fixed for 20 min in fresh 4% paraformaldehyde in PBS and subsequently washed five times with PBS. This incubation was followed by permeabilization and blocking as described above. Primary antibodies were added to the chambers at appropriate dilutions in blocking buffer [anti-RPTPm antibody SK-15 (1:1000 dilution of ascites)], g-catenin (1:2000), b-catenin (1:3000), anti-vWF (Sigma Co.) (1:500), and anti-VE-cadherin (1:500) (Chemicon International Inc., Temecula, CA) for 1 h, followed by three washes with PBS and incubation with the appropriate secondary antibodies, either goat anti-rabbit IgG coupled to FITC (1:200) or donkey anti-mouse IgG coupled to Cy3 (1:500) (Jackson Immunoresearch Laboratories, West Grove, PA), for 30 min. Excess secondary antibodies were removed by washing five times with PBS, and the slides were mounted in Vectashield media (Vector Laboratories, Burlingame, CA). Images were obtained with an Olympus upright microscope. Photographs were taken with Kodak TMAX 400 ASA film at the same exposure time for all pairs of micrographs and were developed and printed under identical conditions. Immunoadsorption experiments with GST alone or GST–RPTPm [16] were performed by preincubating the primary antibodies (three times for 20 min each time) with 10 –20 mg of GST or GST–RPTPm immobilized on a PVDF membrane (Immobilon-P, Millipore Corp., Bedford, MA) at RT before applying them to the tissue sections. Control experiments with the secondary antibody alone were routinely performed and, unless otherwise stated, produced no significant background (data not shown). Immunoblotting. Total cell extracts were prepared from sparse or confluent BAE as follows: The dishes were washed in cold PBS, scraped with a rubber policemen, pelleted in a table-top centrifuge, and ressuspended in water. Aliquots were taken to measure protein using a BCA protein assay kit (Pierce, Rockford, IL) and the remaining portion was lysed directly in Laemmli buffer (62.5 mM Tris–HCl, 2% SDS, 1% b-mercaptoethanol, 10% glycerol, and 0.0025% bromophenol blue) [17]. The lysate was boiled for 5 min, and ;10 mg / 20 ml was subjected to 8% SDS–polyacrylamide gel electrophoresis (SDS– PAGE). Proteins were transferred to Immobilon-P using a semidry blotting apparatus (Bio-Rad, Richmond, CA), membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.1% Tween 20) for 1 h at RT, and the blots were probed with mouse monoclonal anti-RPTPm antibody BK-2 (1:3000 dilution of ascites) in TBST plus 5% milk overnight at 4°C. Unbound antibodies were removed by three 5-min washes in TBST, followed by incubation for 30 min with horseradish peroxidase-coupled antimouse antibodies (Amersham, Little Chalfont, Buckinghamshire, UK) at 1:3000 dilution. Following washing, bound antibodies were visualized by enhanced chemiluminescence (ECL) using Hyperfilm
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FIG. 1. Localization of RPTPm in the rat vascular endothelium. Frozen sections from rat carotid artery (A) and aortic vasa vasorum (B) were prepared and immunostained with antibody SK-15 as described under Materials and Methods. Specificity of RPTPm staining in rat left ventricle sections is shown by comparison of SK-15 immunostaining following (C) preincubation with GST alone or (D) preincubation with GST–C-terminal RPTPm. Arrows (A, B) show staining at sites of cell– cell junction. Note that staining of endothelial cells (small arrows in C) and intercalated discs (larger arrows in C) is blocked upon preincubation of SK15 with its immunogen (D). L, lumen; M, muscularis; E, endothelium; C, capillary. Bar 5 10 mm.
(Amersham). To verify proper fractionation and transfer of proteins, membranes were stained with 0.25% Coomassie brilliant blue R-250 (Sigma Co.) for 2 min, followed by a 10-min wash with 25% methanol/7% acetic acid solution [18].
RESULTS
Expression of RPTPm in the Vascular Tree Expression of RPTPm in various tissues was assessed by indirect immunofluorescence using a monoclonal antibody (SK-15) directed against its first (i.e., more N-terminal) PTP domain. The generation, preparation, and characterization of this antibody have been described previously [7, 10, 16]. It is monospecific for detecting RPTPm in a variety of endothelial cells of different origin (including different species). Expres-
sion was detected in all tissues sampled, but within most immunoreactive RPTPm was restricted to the vascular endothelium (Table 1; see figures below). In the vasculature, RPTPm immunostaining was prominent in large and small arteries (Fig. 1A), their immediate tributaries to arterioles, and the vasa vasorum (Fig. 1B). SK-15 immunoreactivity was unaffected by preincubation with GST alone (Fig. 1C), but immunoreactivity was completely eliminated by preincubation with a GST–RPTPm fusion protein (Fig. 1D), confirming the specificity of our immunoreagents. Notably, RPTPm expression was undetectable in most capillary beds (e.g., kidney, spleen, liver, adrenal), with the exception of those in heart, brain, and skeletal muscle (Figs. 2A and 2B and data not shown). The expression of RPTPm in these capillary beds may reflect particular features
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herins in lysates from cultured cells and primary tissues. In addition, previous studies have shown that RPTPm expression in tissue culture cells is restricted to cell– cell junctions, where it colocalizes with cadherin– catenin complexes [9, 10]. We noted that RPTPm immunoreactivity in tissue sections was restricted to endothelial cell junctions (Figs. 1–5). Consistent with the previous observations in cultured cells, RPTPm also colocalized with b-catenin, g-catenin/plakoglobin, and cadherins, as revealed by double indirect immunofluorescence studies (Figs. 2A and 2B; see also Figs. 7A and 7B and data not shown). RPTPm Expression outside the Vascular System In addition to cardiac arteries and capillaries (Fig. 1), strong RPTPm immunoreactivity was observed in intercalated discs of the heart (Figs. 1C, 3A, and 6A), which do not stain anti-vWF (Figs. 3B and 6B). Interestingly, colocalization of RPTPm and components of
FIG. 2. RPTPm and g-catenin colocalize in endothelial cells. Double immunofluorescence of RPTPm (A) and g-catenin (B) in the vasculature of rat skeletal muscle. Note the colocalization of staining with both antibodies at areas of cell– cell contact (arrows) in an arteriole. L, lumen. Bar 5 10 mm.
of their junctions (see Discussion). RPTPm expression also was undetectable in the endocardium. In the same sections, endocardial cells were easily visualized by anti-von Willebrand factor (vWF) antibodies in double indirect immunofluorescence experiments (Figs. 3A and 3B). Interestingly, the endocardium and other endothelial cells are ontogenetically distinct (see Discussion). RPTPm expression was undetectable in most veins, although immunoreactivity was observed in some larger veins (Figs. 4A and 4B and data not shown). However, even in these, PTPm immunoreactivity was much less intense than in arteries of similar caliber (Figs. 4A and 4B). These differences between arterial and venous RPTPm and vWF immunoreactivities were recapitulated in human umbilical cords (Figs. 5A–5D) despite the lack of difference of adherens junctions as determined by VE-cadherin staining in consecutive sections of the same cords (Figs. 5E and 5F) (see below). RPTPm co-immunoprecipitates with several cad-
FIG. 3. Lack of detectable expression of RPTPm in the endocardium. Frozen sections of the rat ventricular free wall were immunostained for RPTPm and vWF as described under Materials and Methods. Note the absence of RPTPm staining (A) in the endocardium (arrows), compared with the intense staining for vWF (B). i, intercalated discs; C, capillaries. Bar 5 10 mm.
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FIG. 4. Heterogeneity of RPTPm expression in different vascular beds. Immunostaining for RPTPm (A) and vWF (B) in the vasculature of the rat adrenal capsule and its surrounding fat. Note the dramatic difference in staining intensity between RPTPm (A) in endothelial cells in the arteriolar (a) and venous (v) trees, compared to vWF (B). M, medial muscular layer. Bar 5 100 mm.
cadherin/catenin complexes was also observed in these structures (data not shown). RPTPm was also detected at low levels at sites of cell– cell contact in the bronchial epithelium (Figs. 7A and 7B) and in the biliary tract epithelium (data not shown).
RPTPm Expression in Endothelial Cell Cultures Previous studies have examined endogenous RPTPm protein levels in Mv1 Lu cells [9] and heterologously expressed RPTPm in a fibroblast cell line [11]. Our data
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FIG. 5. Lack of difference in VE-cadherin-containing adherens junctions despite marked differences of RPTPm expression in umbilical cord arterial and venous circulations. Frozen sections of a human umbilical cord were immunostained for RPTPm (A, C) and vWF (B, D) (double labeling) in the umbilical vein (B, D) and artery (A, C) and compared with VE-cadherin staining in umbilical artery (E) and vein (F). Note the dramatic difference in staining intensity between RPTPm in endothelial cells in the artery (C) and vein (D) compared with a consecutive section stained with VE-cadherin [artery (E) and vein (D)]. L, lumen; M, muscular layer; endothelium, arrows.
indicate that RPTPm expression in vivo is largely restricted to endothelial cells. Therefore, we monitored RPTPm expression in primary ex vivo endothelial cell cultures. In subconfluent cultures, little RPTPm could be detected at the cell surface, with weak expression primarily confined to a perinuclear region that most
likely represents the Golgi apparatus (Fig. 8A). Consistent with the in vivo studies, RPTPm expression was found at sites of cell– cell junction in confluent BAE cultures (Fig. 8B). Corroborating these immunofluorescence results, immunoblotting experiments confirmed that total RPTPm protein levels are upregulated by cell
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rens junctions between umbilical vein and umbilical arteries as determined by VE-cadherin staining. DISCUSSION
Endothelial cells perform multiple important functions. They are essential for maintaining normal homeostasis, for responding appropriately to inflammatory stimuli, and for controlling endothelial cell permeability to macromolecules [20 –23]. These processes require that proper endothelial junctions be dynamically maintained. Earlier work has shown that signaling via PTKs such as the VEGF receptor can modify vascular permeability [24]. However, the role of specific PTPs in regulating these processes has remained obscure. In this study, we have identified RPTPm as a PTP expressed almost exclusively at endothelial cell junctions in vivo. We find that RPTPm expression is variable in different endothelial cell beds,
FIG. 6. RPTPm expression in intercalated discs. Frozen sections from the rat left ventricle were prepared and immunostained for RPTPm expression (A) and vWF (B), as described under Materials and Methods. Note the staining of intercalated discs (arrows) by RPTPm antibodies, which is not observed with vWF antibodies. C, capillaries. Bar 5 10 mm.
density (Fig. 8C), establishing a direct relationship between cell density, RPTPm protein levels, and RPTPm surface expression in endothelial cells. Note that in endothelial cells, RPTPm undergoes correct processing, generating the mature proteolytically cleaved form, as well as the immature precursor [19], whereas cleavage does not occur detectably in transiently transfected COS cells. Finally, when BAE cells are left confluent for several days, spontaneous sprouting develops, resulting in the formation of a three-dimensional network of tubular structures. As expected from the above studies, RPTPm staining was located predominantly in the cell– cell contacts of these cord-like structures (Fig. 9), reproducing the localization observed in intact vessels. In umbilical cords this heterogeneity is maintained, i.e., despite the presence of venous blood in the arterial tree and arterial blood in the vein (pO 2 levels). In addition, there was no appreciable difference in adhe-
FIG. 7. RPTPm expression and colocalization with catenins in the bronchial epithelium. Double immunofluorescence showing the localization of RPTPm (A) and b-catenin (B) in the rat bronchial epithelium. Note the presence of RPTPm in the bronchi epithelium (arrows in A) which colocalizes with b-catenin (arrows in B). L, lumen; SM, smooth muscle. Bar 5 10 mm.
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Moreover, we find that the RPTPm protein expression pattern only partly overlaps the pattern of expression of its mRNA, arguing that RPTPm expression is regulated by posttranslational as well as transcriptional mechanisms. Our data suggest that RPTPm may function in transducing signals from cell– cell junctions in endothelial cells. Alternatively, or in addition, RPTPm may help control the integrity of endothelial cell junctions. In this regard, preliminary studies indicate that VEGF stimulation causes redistribution of RPTPm expression (R.D.V. and N.K.T., unpublished observations). RPTPm is expressed in the endothelium of largecaliber arteries and in selected capillary beds, providing yet another example of endothelial cell heterogeneity. Multiple studies have shown that endothelial cells in different anatomical locations express distinct molecular markers [22]. In some cases, this heterogeneity
FIG. 8. Modulation of RPTPm expression and compartmentalization in endothelial cell cultures. Bovine aortic endothelial cells cultured on eight-well glass chamber slides either sparsely (A) or at high density (confluent) (B) were immunostained for RPTPm expression. Note the variation in intensity of RPTPm staining and its localization with changes in cell density. (C) Immunoblot of BAE total cell lysates probed with anti-RPTPm monoclonal antibody BK-2. (Left) Two independent experiments comparing BAE under confluent (c) versus sparse (s) conditions. For quantitation, one-half (c/2) and one-quarter (c/4) of the amount of protein from a confluent culture were also loaded. Note the difference in RPTPm protein levels in sparse (s) versus confluent (c) cells. (Right) Effect of increasing culture time on RPTPm expression. BAE were seeded at approximately 50% confluency, (50%) and allowed to grow for 2, 5, or 10 days, as indicated, before determination of RPTPm expression levels. COS cells transiently transfected with RPTPm serve as positive controls for immunoblotting. The size of RPTPm in transfected COS cells is different because of incomplete maturation. N, nuclei; arrows, cell– cell contacts; c, confluent; s, sparse.
with expression much more prominent in large and small arteries than in veins and capillaries, and that its surface expression is regulated by cell density.
FIG. 9. Localization of RPTPm in BAE undergoing tube formation ex vivo. (A) Localization of RPTPm at sites of cell– cell contacts (arrows) in cord-like structures of BAE induced to undergo tube formation (see Materials and Methods for details). (B) Phase contrast of A. Bar 5 10 mm.
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correlates with morphological differences that, in turn, correlate with differences in vascular permeability. For example, the continuous endothelium lining the central nervous system expresses distinct markers compared with the discontinuous endothelium found at other anatomical sites [22]. RPTPm protein expression is highest in the continuous endothelium present in tissues such as skeletal muscle, heart, and lungs. Conversely, little RPTPm protein is expressed in organs with discontinuous (i.e., fenestrated) endothelium, such as the adrenals, gastrointestinal tract, or renal glomeruli, and RPTPm is undetectable in leaky endothelial cell beds, such as those found in the venous circulation of the liver and spleen. This strong correlation between RPTPm protein expression and the presence of endothelial junctions (i.e., cell– cell contact) is consistent with the observation that RPTPm protein levels and surface expression are highly regulated by cell– cell contact (i.e., cell density) in ex vivo cultured cells (Fig. 8). However, the mere presence of cadherin-containing junctions is not sufficient to account for RPTPm expression. In umbilical cord vessels (arteries and veins), the expression of the adherens junction endothelial cell marker (VE-cadherin) is quite similar, yet differences in RPTPm immunoreactivity were dramatic in the arterial compared to venous endothelium and other vascular territories. It should also be pointed out that in the umbilical cord venous blood circulates through umbilical arteries whereas arterial blood circulates through umbilical veins. Therefore, it seems that differences in blood pH and oxygenation alone can not determine whether or not RPTPm is expressed at the EC surface. A recent study monitored RPTPm mRNA expression by in situ hybridization during early mouse development [14]. RPTPm mRNA expression is weak and diffuse at stages 8.5 and 9.5. During embryonic days 12.5 and 14.5, transcription in developing blood vessels is very prominent; similar results were obtained in rat embryos at embryonic day 13.5 [13]. Expression is also observed at some extravascular sites during development, such as the brain parenchyma, particularly the olfactory bulb and cerebellum. By embryonic day 18.5, RPTPm expression was no longer generalized throughout the vasculature, with expression restricted to the heart, kidneys, brown fat in the neck, and, most intensely, lungs. Our work defining the pattern of RPTPm protein expression in adult rats reveals both similarities and some differences with the in situ hybridization studies. We find that RPTPm is expressed in the vasculature of many organs, but shows a particular preference for the arterial bed and capillary territories of the heart, skeletal muscle, and brain. Moreover, we detect no expression of RPTPm in the brain parenchyma, although we did find expression in two
sites (bronchial epithelium and biliary ducts) not observed in the earlier work. It is not clear whether the differences between RPTPm mRNA expression in E18.5 mice and RPTPm protein expression in adult rats reflect further transcriptional regulation of RPTPm during later mouse development, additional, posttranslational mechanisms of regulating RPTPm expression, and/or species-specific differences. Although RPTPm protein is highly expressed in nearly all endothelial cells that form cell junctions, endocardial cells provide a notable exception. Interestingly, previous studies have shown that endocardial cells have a distinct ontogenetic origin from those of nearly all other endothelial cells, deriving from the more axial mesoderm [22]. Since cell– cell junctions are abundant in the endocardium, the absence of RPTPm expression in these cells presumably reflects transcriptional or posttranscriptional regulation, perhaps due to the distinct developmental origin of endocardial cells. Previous work showed that endogenous RPTPm is expressed at points of cell– cell junction in Mv1 Lu epithelial cells [9]. Since we find that bronchial epithelial cells express RPTPm (Fig. 7A). Mv1 Lu cells, which were established from a mink lung, most likely derive from cells of similar origin [28]. Our data support a role for RPTPm in regulating cell– cell junctions in the bronchial epithelium, most likely through interactions with cadherin/catenin complexes. Given the known function of tyrosyl phosphorylation in the dynamic regulation of cell– cell junctions, RPTPm is likely to play an important role in controlling junctional integrity in response to various stimuli. Future studies will be required to define the precise signaling pathways and targets of RPTPm in endothelial and epithelial cell junctions. This work was supported by P01 HL56993-01 (C.B. and B.G.N.), HL-46716 (F.W.S.), and GM 55989 (N.K.T.).
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