Division of Gastroenterology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6145
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ABSTRACT |
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The mechanisms controlling tyrosine phosphorylation of cellular proteins are important in the regulation of many cellular processes, including development and differentiation. Protein tyrosine phosphatases (PTPases) may be as important as protein tyrosine kinases (PTKs) in these processes. PRL-1 is a distinct PTPase originally identified as an immediate-early gene in liver regeneration whose expression is associated with growth in some tissues but with differentiation in others. We now demonstrate that the PRL-1 protein is expressed during development in a number of digestive epithelial tissues. It is expressed at variable time points in the developing intestine, but its expression is limited to the developing villus enterocytes. In the gastric epithelium, PRL-1 expression in the adult is restricted to zymogen cells. PRL-1 is also expressed in the developing liver and esophagus and in the epithelia of the kidney and lung. In each of these contexts, the expression of PRL-1 is associated with terminal differentiation, suggesting that it may play a role in this important developmental process.
tyrosine phosphatase; differentiation; signal transduction
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INTRODUCTION |
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INSIGHT INTO the molecular and cellular mechanisms controlling the development and differentiation of digestive and other epithelial tissues has been increasing in recent years. However, the role of tyrosine phosphorylation and attendant signal transduction pathways in these processes requires greater elucidation. Because tyrosine phosphorylation is involved in the regulation of many cell functions, playing a critical role in the regulation of gene expression, enzymatic activity, cell cycle progression, and cellular transport and motility (12, 13, 18), it is likely that roles exist in the regulation of tissue development and differentiation as well.
Protein tyrosine phosphatases (PTPases) are as important as tyrosine kinases in regulation of important tyrosine phosphorylation signaling pathways (9, 33, 36, 40). Recent experiments indicate that some PTPases play significant roles in vertebrate development. Sokol and colleagues (34) showed that interruption of SHP2 PTPase signaling by overexpression of a dominant mutant PTPase resulted in severe posterior truncation mutations. Several receptor PTPases are of critical importance in the fetal brain, influencing cell-cell adhesion, neurite growth, axon guidance, and target cell recognition (30, 32). The receptor PTPase LAR-PTP2 was found to be highly expressed in fetal lung only at the particular point of maximal tissue differentiation when terminal bronchioles and alveolar sacs are being formed (28). In situ hybridization experiments have demonstrated that the MKP-1 PTPase is expressed in a tissue-restricted and temporal pattern during mouse development. Significant levels were found early in gut development, with mRNA detected at 10.5 days, whereas expression later in development was found to be limited to villus enterocytes (3).
We first identified the nonreceptor PTPase PRL-1 as an immediate-early growth-response gene in regenerating liver and mitogen-treated 3T3 fibroblasts (6, 23). In addition to regenerating liver, PRL-1 is expressed in skeletal muscle and brain, two terminally differentiated tissues (6). PRL-1 is also expressed significantly in intestinal epithelia, and, in contrast to the expression pattern of PRL-1 in liver, its expression is associated with cellular differentiation in the intestine. Specifically, PRL-1 is expressed in villus but not crypt enterocytes and only in differentiated Caco-2 colon carcinoma cells (8). These results suggest that PRL-1 may play different roles depending on tissue context.
To explore these issues further, we determined the expression pattern of PRL-1 during digestive tissue development. We found that PRL-1 is expressed at a number of different time points in the developing small and large intestine and that its expression in this context is associated with enterocyte differentiation. Additionally, PRL-1 is expressed in the gastric epithelium, with restriction to developing and adult zymogen cells. Furthermore, PRL-1 is expressed in the developing esophagus and liver and in the developing and adult kidney and lung.
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MATERIALS AND METHODS |
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Animals. Adult and postnatal tissue samples were harvested from Sprague-Dawley rats (160-200 g; Charles River). For developmental time points, timed pregnant female Sprague-Dawley rats were obtained (Charles River). At the indicated time points, the animals were killed by cervical dislocation and the abdomen was opened. The uterine horns were identified, the cervical, oviduct, and vaginal linkages were cut, and the uterine horns were lifted clear, placed in a petri dish in PBS, and incised on the antimesoatrial side (11).
Immunohistochemistry. Tissues were removed and fixed overnight at 4°C in 4% paraformaldehyde in PBS, pH 7.4. Paraffin sections (6 µm thick) were prepared at room temperature, deparaffinized with xylene, and rehydrated in ethanol. After endogenous alkaline phosphatase activity was quenched by treating the slides in 0.2 N HCl for 15 min, the sections were blocked with avidin followed by biotin (Vector) and then further treated with protein blocking agent (Immunotech). The sections were then incubated overnight at 4°C with affinity-purified rabbit polyclonal anti-PRL-1 antibody (0.17 µg/ml) or an equivalent concentration of nonimmune rabbit IgG (Sigma) as a control. The anti-PRL-1 antibody is the same one we have used previously (6, 8). The integrity of this antibody was previously demonstrated by detection of transfected PRL-1 by Western blot and immunofluorescence studies and by the absence of staining by anti-PRL-1-depleted sera prepared from the flow-through of a PRL-1 affinity column (6). In select cases, tissues were stained with rabbit polyclonal anti-human intrinsic factor antibody (a generous gift of Dr. David Alpers). Sections were then extensively washed with PBS and incubated with biotinylated anti-rabbit antibody (Biostain) at 37°C for 30 min, followed by a 30-min incubation with avidin-biotin complex reagent (Biostain). The sections were washed in PBS and equilibrated in SMT buffer (100 mM NaCl, 50 mM MgCl2, 0.01% Tween, and 10 mM Tris, pH 9.5). The color development was performed at room temperature in SMT buffer containing nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-indolyl phosphate (BCIP) (Boehringer Mannheim). Light counterstaining was done with 2% neutral red for 2 min, and the sections were then dehydrated and mounted.
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RESULTS |
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PRL-1 is expressed at multiple time points in developing intestine
in association with epithelial differentiation.
In Fig. 1A, serial sections of
embryonic day (E)18 foregut are stained with anti-PRL-1 antibody (Fig.
1A, i and ii) and control sera (Fig.
1A, iii). In the anti-PRL-1-stained specimen,
nuclei are prominently stained in some protovillus enterocytes. There is some cytoplasmic staining in other enterocytes as well. At this
stage of development, the intestinal crypts are not completely formed,
but it can be observed that there is no PRL-1 staining in the base of
the villi. Figure 1B shows representative sections of E18
midgut (Fig. 1B, i and ii) stained
with anti-PRL-1 antibodies or control sera (Fig. 1B,
iii). PRL-1 staining areas are seen in the more distal
enterocytes of the protovilli but not in the bases. Finally, a section
from the E18 hindgut (Fig. 1C) demonstrates an absence of
anti-PRL-1 staining. In contrast to the developing small intestine, it
appears that PRL-1 expression begins later in the developing colon. We
found that intestinal sections from E14 rats did not stain for PRL-1 in
any segment, either small intestine or colon (data not shown). At this
time point, villus buds have just begun to form from the preceding
pseudostratified intestinal epithelium. This finding would appear to
indicate that PRL-1 is not absolutely required for villus formation
but, rather, it may play a role in the differentiation of these cells
into mature enterocytes.
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PRL-1 is expressed in zymogen cells of the stomach but not in
parietal or mucus cells.
The cells of the gastric glands in the adult stomach are arranged in a
distinct architecture, and their characteristic morphological features
permit them to be distinguished from one another (20). The
zymogen cells can be distinguished by their granularity and by their
location within the gland (16, 17, 20, 21). Figure 3A shows a representative
section of adult stomach stained with the anti-PRL-1 antibody. A
higher-power view is shown in Fig. 3B. Anti-PRL-1 staining
is present only in the nuclei of granular cells in the base of the
gastric glands, which are likely to be zymogen cells. However, PRL-1
expression is not observed in the well-differentiated parietal or mucus
pit cells. To verify the identification of zymogen cells, Fig.
3C shows a serial section stained with anti-intrinsic factor
antibody. This antibody has previously been established to stain only
gastric zymogen cells in the rat (2, 31).
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PRL-1 is expressed in a subset of cells in developing
esophagus and liver.
Figure 4A, i,
shows a representative section of E14 esophagus stained for PRL-1. At
this point in development, the cells lining the lumen in this organ are
still columnar, having not yet assumed their eventual squamous
morphology (1). Anti-PRL-1 staining is evident in the
nuclei of some of these cells. No staining was noted in serial sections
stained with control antisera (data not shown). At later developmental
time points, and in the adult, when the esophageal epithelium has
changed from columnar to squamous, we found no evidence of PRL-1
expression in this organ (data not shown). Thus it appears that the
expression of PRL-1 in the developing gut may be restricted to cells
with a columnar phenotype and is lost in the esophagus when the
columnar epithelium is replaced by the squamous type.
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PRL-1 is significantly expressed in kidney and lung both in adult
and during development.
In addition to expression in digestive tissues, staining with the
anti-PRL-1 antibody was present in other epithelial tissues, both
during development and in the adult. Figure
5 shows the pattern of anti-PRL-1
staining in the kidney. In the adult rat (Fig. 5A), anti-PRL-1 staining is seen to be present only in a small proportion of
cells in the renal cortex. A higher-power view (Fig. 5B)
shows that these are likely to be proximal tubule cells because of
their brush border. The glomeruli are clearly not stained by the
anti-PRL-1 antibody. PRL-1 appears to be localized to both the nuclei
and the cytoplasm of these cells. This staining pattern is not seen in
a section stained with control sera (Fig. 5C). A similar
pattern is seen during renal development. Figure 5D shows a
representative section of kidney from an E18 animal. PRL-1 staining is
seen in both the nuclei and the cytoplasm of the tubular cells at this time point.
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DISCUSSION |
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Our findings are consistent with the supposition that the PRL-1 PTPase may be important in the development and maintenance of differentiating epithelial tissues. It is expressed in specific terminally differentiated cells in a number of digestive organs, including the enterocytes of the small intestinal villi and colonic surface and the zymogen cells of the stomach. In the fetal counterparts of these adult tissues, as well as in the fetal (but not adult) esophagus and liver, PRL-1 is also expressed early in epithelial development, suggesting that it may play a role in guiding the development process in these tissues. In the gut, regional differences in the magnitude and timing of PRL-1 expression can be discerned, with higher and earlier levels detected in the proximal small intestine than in more distal segments and the colon. PRL-1 is also expressed, during development and in the adult, in the epithelial cells lining the pulmonary bronchioles and in the proximal tubular cells of the kidney.
In a number of mature tissues, including the epithelia of the small intestine, stomach, and lung, PRL-1 expression appears to be mostly or exclusively nuclear. However, a pattern of staining in both the nucleus and the cytoplasm was noted in the epithelia of the colon and renal tubules. In addition, a number of tissues that display a nuclear pattern of localization for PRL-1 in the adult show evidence of some cytoplasmic expression during the tissue's development. In general, it appears that the expression of PRL-1 becomes more consistently nuclear, as well as more cell type restricted, as development progresses. The function of PRL-1 may be governed in part by its subcellular localization. In this regard, it is interesting to note that PRL-1 contains a carboxy-terminal CAAX motif, which is a marker for protein prenylation. The last four amino acids CCIQ in PRL-1 point toward farnesylation as the most likely modification. It has been shown that PRL-1 can be farnesylated in vitro and is at least partly farnesylated in vivo (4). Because the main roles of protein prenylation are attachment to cellular membranes and protein-protein interactions (5, 39), it is possible that control of this modification may underlie the specificity of the function of PRL-1 in a given cell type.
It is important to note that two PRL-1 homologues, PRL-2 and PRL-3, have been identified (4, 38). PRL-2 has been cloned and has been found to be expressed in a number of tissues, with particularly high mRNA levels in skeletal muscle. PRL-3 was found in an expressed sequence tag database search for PRL-1 homologues and has not been fully cloned. Both proteins are predicted to be highly homologous to PRL-1, with little homology to other PTPases outside of the active site (24, 25, 38). Specific antibodies are not available for either of the two homologues. Because there is a significant degree of homology, it is possible that some of the results noted here could in fact reflect expression of PRL-2 or PRL-3 instead of PRL-1. However, several lines of evidence suggest that this is not the case. First, we have obtained a PRL-2 clone (generous gift of Dr. Gilbert Lenoir) and expressed it in bacteria. Our PRL-1 antibody does not cross-react with this protein on Western blots (R. Diamond, unpublished data). Second, PRL-1 mRNA expression has been identified by in situ hybridization in developing lung, kidney, and midgut tissues (29). The current work is the first to examine the expression pattern of the PRL-1 protein in these tissues. Because the in situ hybridization results, which are more specific for a particular homologue, agree with our data on PRL-1 protein expression, it is likely that our data accurately represent PRL-1 expression and not that of its cellular homologues. Third, Northern blot data do not indicate expression of PRL-3 in kidney (38), as we have shown here, whereas the Northern blot results do indicate expression of PRL-3 in heart (38), a tissue we found not to be stained by the PRL-1 antibody (data not shown). Fourth, anti-PRL-1 staining can be specifically blocked with bacterially expressed PRL-1 protein (37). Finally, we have previously documented the specificity of our anti-PRL-1 antibody with Western blot and immunofluorescence data after PRL-1 overexpression and have shown the abrogation of these staining patterns after anti-PRL-1 depletion of the antisera by affinity chromatography (6). In short, although we cannot definitively exclude that some of our data may reflect expression of PRL-1 homologues, this possibility appears unlikely.
Because PRL-1 is a tyrosine phosphatase, a complete understanding of its cellular function awaits the precise identification of its natural substrates and other interacting proteins. In this regard, we have recently identified a novel transcription factor that interacts with PRL-1 with the use of the yeast two-hybrid system (Diamond et al., unpublished observations). PRL-1 might positively or negatively regulate the DNA binding or transactivation of this transcription factor. If the primary role of PRL-1 is in transcriptional regulation, its localization to the cytoplasm under some circumstances could represent modulation of its activity by sequestration. Ultimately, experiments involving forced under- and overexpression of PRL-1 in specific cells and tissues will be necessary to investigate its role in bringing about the phenotypes with which its expression is associated. Such experiments are currently underway in intestinal cell lines in our laboratory.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Anil K. Rustgi for thoughtful comments and suggestions on this manuscript.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant RO1-DK-52216 (R. H. Diamond) and by the Morphology Core of the University of Pennsylvania NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases P30-DK-50306.
Address for reprint requests and other correspondence: R. H. Diamond, GI Division, Univ. of Pennsylvania School of Medicine, 664 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6145 (E-mail: diamondr{at}mail.med.upenn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 30 November 1999; accepted in final form 12 April 2000.
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