ARTICLE |
Correspondence to: Patricia Andrade-Gordon, R.W. Johnson Pharmaceutical Research Institute, Welsh and McKean Roads, Spring House, PA 19477.
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Summary |
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PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2. (J Histochem Cytochem 46:157164, 1998)
Key Words: PAR-2 (protease activated, receptor 2), PAR-1 (thrombin receptor), human, immunohistochemistry, flow cytometry
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Introduction |
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The DISCOVERY of protease-activated receptors (PARs) has suggested the possibility of a novel family of G-protein-coupled receptors that undergo a unique mechanism of activation via proteolytic cleavage of the amino terminus, subsequently exposing a tethered peptide ligand (
The tissue distribution and hence the function of PAR-2 have only recently begun to be elucidated. Expression patterns derived from Northern analysis indicated an abundance of PAR-2 mRNA in kidney, stomach, pancreas, liver, colon, and small intestine (
We used this antibody to immunophenotype a series of normal human tissues. In addition, an antibody to the human PAR-1 was used for tissue-specific comparisons. We report significant PAR-2 immunolocalization in epithelial cells and smooth muscle of vascular and nonvascular origin. We also provide in vivo evidence for PAR-2 expression in the brain, supporting a recent report demonstrating PAR-2 expression and function in rat hippocampal cultures (
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Materials and Methods |
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Preparation of Antibodies
Peptides were linked through a terminal cysteine to maleide-activated KLH (Pierce; Rockford, IL) according to the manufacturer's instructions. For antibody generation to human PAR-2, a peptide containing the sequence SLIGKVDGTSHVTGKGVC (residues 3753) was used. This sequence is present in both cleaved and uncleaved receptors. Unlinked peptide was removed by dialyzing against PBS (50-kD cutoff). New Zealand White rabbits were immunized by SC injection (multiple points) with 0.1 mg of KLH peptide in PBS emulsified with adjuvant in accordance with institutional guidelines. Injections were performed at 2-week intervals and the animals were bled 10 days after boosting. Anti-peptide antibody titers were measured by indirect ELISA performed on peptide-coated plates. To affinity-purify the antibody, the peptide was coupled to iodoacetylagarose beads (Sulfolink; Pierce) according to the manufacturer's instructions. The specific antibody was then eluted from the gel using 0.1 M glycine, pH 2.4, and the eluted material was immediately neutralized with Tris base before being dialyzed against PBS. This antibody has been named PAR-2C. It was used at a titer of 1 µg/ml for immunohistochemical staining.
WEDE15, an antibody specific to PAR-1, is a previously described IgG1 monoclonal antibody (MAb) produced in mice immunized with PAR-1 peptide KYEPFWEDEEKNES, corresponding to residues 5164 of the human PAR-1 sequence (
Expression of PAR-1 and PAR-2 in Sf9 Cells
A 430-BP fragment of the murine PAR-2 cDNA was used as a probe for screening a human keratinocyte cDNA library (Clontech; Palo Alto, CA). A full-length clone of human PAR-2 was identified. Restriction sites were introduced immediately before and after the coding region of this clone by PCR and the cDNA was cloned in the Baculovirus expression vector pAcMP2. A human PAR-1 cDNA was similarly modified and cloned in the above vector. Each expression construct was co-transfected with baculogold AcNPV DNA (PharMingen; San Diego, CA) in Sf9 cells according to the manufacturer's instructions. Recombinant viral clones were amplified and titrated by infection of Sf9 cells.
Analysis of PAR-1 and PAR-2 Expression by Flow Cytometry
Monolayer cultures of Sf9 cells were infected with PAR-1- or PAR-2-encoding virus at a multiplicity of infection of 2 and were incubated for 72 hr at 27C. Cells were dislodged, washed once in PBS, and 5 x 105 cells were resuspended in staining buffer (PBS with 1% BSA and 0.02% sodium azide) with the PAR-2C or WEDE15 antibody at 10 µg/ml. Cells were stained for 1.5 hr on ice, washed with ice-cold PBS, and resuspended in staining buffer containing 5 µg/ml FITC-conjugated goat anti-rabbit (PAR-2C-stained cells) or 5 µg/ml FITC-conjugated goat anti-mouse antibody (WEDE15 stained cells) (Cappel; Durham, NC), and 10 µg/ml propidium iodide. The stained cells were analyzed on a FACScan flow cytometer (Becton Dickinson; Mountain View, CA). The live cells were gated.
Cell Culture
Human neonatal epidermal keratinocytes and human dermal fibroblasts (AG1523) were cultured as previously described (
Human Platelets
Human platelet concentrate (105 platelets/µl) was purchased from Biological Specialty Corporation (Colmar, PA). Platelets were collected from drug-free human donors by plasmaphoresis on a Cobe Spectra using ACD (acid citrate dextrose solution). Human platelets were routinely fixed in 4% paraformaldehyde for 2 hr at RT and then processed for paraffin embedding.
Immunohistochemistry
Human multitissue blocks were purchased from Biomeda (Foster City, CA) and Dako (Carpenteria, CA). Tissue sections were mounted on slides, deparaffinized, and hydrated. Slides were immersed in Target reagent (Dako) heated two times for 3 min at high power in an 800-W commercial microwave. After the slides cooled, the endogenous peroxidase was blocked by 3.0% H2O2 for 10 min. Tissue slides were processed through an avidinbiotin blocking system according to the manufacturer's instructions (Vector Labs; Burlingame, CA) and then placed in PBS. Platelets were embedded in paraffin, cut onto slides, and then routinely dewaxed, hydrated, and then placed in PBS.
All reagent incubations and washes were performed at RT. Normal blocking serum was then placed on all slides for 10 min (Vector Labs). After brief rinsing in PBS, primary antibodies were placed on the slides for 30 min (Table 1). The slides were washed and then the biotinylated secondary antibodies, goat anti-rabbit (PAb) or horse anti-mouse (MAb) were placed on the tissue sections for 30 min (Vector Labs). After rinsing in PBS, the avidinbiotinHRP complex reagent (Vector Labs) was added for 30 min. The slides were washed and treated with the chromogen 3,3'-diaminobenzidine (Biomeda; Foster City, CA) two times for 5 min and rinsed in dH2O, counterstained in Mayer's hematoxylin, dehydrated, and then coverslipped. The slides were visualized and photographed using an Olympus BX50 light microscope.
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Controls were run for each tissue. Negative controls included replacement of the primary antibody with the antibody diluent (Zymed Laboratories; San Francisco, CA) and the use of the same species isotype nonimmunize serum. Specificity of the antibodies for PAR-2 and PAR-1 was confirmed by preincubation overnight at 4C with their respective antigen in a 20-fold molar excess of antigen to antibody. In addition, the PAR-2C antibody was preincubated with peptide corresponding to the PAR-1 amino terminal sequence SFLLRNPNDKYEPF relative to the antigen sequence used to generate PAR-2C. Positive control antibodies included relevant cellular markers (Table 1).
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Results |
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Specificity of PAR-2C
The specificity of PAR-2C for PAR-2 was first examined using cells expressing recombinant human PAR-2. Flow cytometry analyses performed on Sf9 cells infected with recombinant baculoviruses containing either human PAR-2 or PAR-1 cDNA demonstrated that PAR-2C was selective for PAR-2 and did not bind to PAR-1 (Figure 1). In addition, the MAb WEDE15 recognized PAR-1 and did not crossreact with PAR-2.
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Negative immunohistochemical controls were devoid of any detectable immunolabeling. Preincubation of PAR-2C and the PAR-2 antigen also resulted in absence of immunolabeling. Furthermore, preincubation of PAR-2C with the PAR-1 antigen did not affect PAR-2C immunolabeling, providing additional evidence that PAR-2C does not crossreact with PAR-1. The positive control antibodies labeled according to the manufacturer's specifications.
To further characterize the specificity of this antibody for endogenous PAR-2, staining patterns were evaluated in human dermal fibroblasts and human epidermal keratinocytes. We have previously demonstrated that both cell types express PAR-1 mRNA and possess functional PAR-1. However, PAR-2 is expressed exclusively in keratinocytes and not in fibroblasts (
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Human platelets represent another cell type that expresses PAR-1 but does not express PAR-2 (
Tissue and Cellular Distribution of PAR-2
The distribution of PAR-2 immunolocalization in human tissues is summarized in Table 2. PAR-2 was abundant in the skin, vasculature, gastrointestinal tract, lung, brain, prostate, and uterus. Overall, PAR-2 exhibited marked expression in most epithelial and smooth muscle cells.
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In the skin, PAR-2 was observed in the basal, spinous, and granular cell layers of the epidermis (Figure 2F). PAR-1 was localized in the basal and spinous cell layers of the epidermis (Figure 2G). However, in contrast to the immunolocalization of PAR-2, PAR-1 was not expressed in the granular layer of the epidermis. In addition, stromal fibroblasts of the dermis were negative for PAR-2 but were positive for PAR-1, consistent with the results in cultured human dermal fibroblasts (data not shown).
PAR-2 was present throughout the vasculature, with strong labeling in both the vascular smooth muscle and the endothelium. As an example, Figure 2H and Figure 2I demonstrate the presence of PAR-2 and smooth muscle actin, respectively, in a human artery from the intestinal submucous plexus. The endothelium of the artery showed intense staining for PAR-2 (arrowheads) and was similarly positive for factor VIII immunolabeling (data not shown). PAR-1 demonstrated similar expression patterns to those of PAR-2 in the vasculature (data not shown).
PAR-2 immunoreactivity was evident in the epithelium of many tissues. Most notable were the tissues of the gastrointestinal tract. In the intestine, PAR-2C labeling extended from the surface to the crypts of the epithelium (Figure 2j). In addition, PAR-2 expression in nonvascular smooth muscle was demonstrated throughout the gastrointestinal tract.
Nonvascular smooth muscle cells as well as stromal myofibroblasts labeled positively for PAR-2 in several tissues in addition to those of the gastrointestinal tract. As shown in Figure 2K, both bronchial epithelium and smooth muscle cells of human lung stained positive for PAR-2. For comparison, Figure 2L demonstrates positive labeling for smooth muscle actin. PAR-2 was also observed in the endothelium and epithelium of the alveolar sacs.
PAR-2 immunoreactivity was also clearly demonstrated in the brain. Neurons showed positive labeling for PAR-2 (Figure 2M) and several astrocytes showed weak immunolabeling (data not shown). Interestingly, not all neurons were positive, indicative of mixed populations of PAR-2-reactive cells. Figure 2N and Figure 2O confirm the immunolabeling of astrocytes (arrowheads) with the antibody to glial fibrillary acidic protein and neurons (arrows) with the antibody neuron-specific enolase, respectively, in the brain.
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Discussion |
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The physiological and/or pathological role of PAR-2 is unknown. Because the endogenous activating protease for PAR-2 has yet to be clearly defined, it has been difficult to identify specific functional roles of PAR-2 in vivo. The aim of this study was to provide a thorough characterization of the tissue-specific cellular localization of PAR-2 protein in vivo. In this report, the tissue distribution of PAR-2 in normal human tissues was described using a polyclonal antibody that selectively recognizes PAR-2. PAR-2 expression was highly expressed in most epithelial cells, including the vascular endothelium. PAR-2 was also abundant in vascular and nonvascular smooth muscle. Expression in stromal cells was tissue-specific. In addition, selective expression in cerebral neurons and astrocytes was noted.
Several reports have implicated PAR-2's involvement in vascular function (
PAR-2 expression was also evident in a variety of nonvascular tissues. Overall, a high correlation of tissue-specific PAR-2 expression in the epithelium and either the respective smooth muscle or the stromal layer was observed. Expression of PAR-2 throughout the gastrointestinal tract, both in the epithelium and the underlying smooth muscle, supports a role for PAR-2 in gastrointestinal function and is consistent with the results of
One notable exception to such a distribution pattern was skin. Expression of PAR-2 only within the epidermis and not the dermis of skin provides a unique example of cell type-specific localization within a single tissue. The expression of PAR-2 within the epidermis suggests that PAR-2 may modulate epidermal structure and function in processes of wound healing, psoriasis, or skin-derived tumors. In vitro studies have indicated that stimulation of PAR-2 in human keratinocytes inhibits both their growth and their differentiation (
The expression of PAR-2 in the brain was a rather unexpected finding, although a recent report supports this observation (
The results of the immunolocalization studies reported here compile a detailed profile of PAR-2 expression in different tissues and suggest a provocative role(s) for this novel receptor. Although this receptor is structurally similar to PAR-1, its physiological function(s) appears to be somewhat different. Clearly, additional studies are needed to define the significance of this receptor in normal or pathophysiological states to place PAR-2 as a distinctive member of this new class of proteolytically activated G-protein-coupled receptors.
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Acknowledgments |
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We thank Christina J. Rogahn for excellent technical assistance, Jack Kauffman for preparation of the human platelets, Drs Charlotte Keenan and Barbara Kulwich for their morphological assistance, and Quality Photo Labs (Voorhees, NJ) for their high-quality photographic reproductions.
Received for publication May 16, 1997; accepted August 14, 1997.
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Literature Cited |
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Al-Ani B, Saifeddine M, Hollenberg MD (1995) Detection of functional receptors for the proteinase-activated-receptor-2-activating polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle. Can J Physiol Pharmacol 73:1203-1207[Medline]
Böhm SK, Kong W, Bromme D, Smeekens SP, Anderson DC, Connolly A, Kahn M, Nelken NA, Coughlin S, Payan DG, Bunnett N (1996) Molecular cloning, expression and potential functions of the human proteinase-activated receptor 2. Biochem J 314:1009-1016[Medline]
Dennington PM, Berndt MC (1994) The thrombin receptor. Clin Exp Pharmacol Physiol 21:349-358[Medline]
Derian CK, Eckardt AJ, AndradeGordon P (1997) Differential regulation of human keratinocyte growth and differentiation by a novel family of protease activated receptors. Cell Growth Differ 8:743-749[Abstract]
Hollenberg MD, Saifeddine M, Al-Ani B (1996) Proteinase-activated receptor-2 in rat aorta: structural requirements for agonist activity of receptor-activating peptides. Am Soc Pharmacol Exp Ther 46:229-233
Hoxie JA, Ahuja M, Belmonte E, Pizarro S, Parton RG, Brass LF (1993) Internalization and recycling of activated thrombin receptors. J Biol Chem 268:13756-13763
Hwa JJ, Ghibaudi L, Williams P, Chintala M, Zhang R, Chatterjee M, Sybertz E (1996) Evidence for the presence of a proteinase-activated receptor distinct from the thrombin receptor in vascular endothelial cells. Circ Res 78:581-588
Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, Coughlin SR (1997) Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386:502-506[Medline]
Magazine HI, King JM, Srivastava KD (1996) Protease activated receptors modulate aortic vascular tone. Int J Cardiol 53(suppl):S75-S80[Medline]
Mirza H, Yatsula V, Bahou WF (1996) The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells. J Clin Invest 97:1705-1714
Molino M, Barnathan ES, Numerof R, Clark J, Dreyer M, Cumashi A, Hoxie J, Schecter N, Woolkalis MJ, Brass LF (1997) Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem 277:4043-4049
Nystedt S, Emilsson K, Larsson A-K, Strombeck B, Sundelin J (1995) Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem 232:84-89[Abstract]
Nystedt S, Emilsson K, Wahlestedt C, Sundelin J (1994) Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 91:9208-9212
Santulli RJ, Derian CK, Darrow AL, Tomko KA, Eckardt AJ, Seiberg M, Scarborough RM, AndradeGordon P (1995) Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc Natl Acad Sci USA 92:9151-9155[Abstract]
SmithSwintosky VL, CheoIsaacs CT, D'Andrea MR, Santulli RJ, Darrow AL, AndradeGordon P (1997) Protease-activated receptor-2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration. J Neurochem in press
Vu TKH, Hung DT, Wheaton VI, Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068[Medline]