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From the Division of Cell Biology,*
Kihara Institute for
Biological Research, Yokohama City University, and Departments of
Surgery
and
Pathology,
Yokohama City University School of
Medicine, Yokohama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trypsin is one of the best characterized serine proteinases. It has long been known that trypsin is produced as a zymogen (trypsinogen) in the acinar cells of the pancreas, is secreted into the duodenum, is activated into the mature form of trypsin by enterokinase, and functions as an essential food-digestive enzyme.4 However, little is known about the distribution and function of trypsin in other normal tissues. To date, four trypsin (or trypsinogen) genes have been characterized in humans: trypsin 1,5 trypsin 2,5 trypsin 3,6 and trypsin 4.7 Trypsins 1, 2, and 3 have been demonstrated as the zymogens in human pancreatic juice.8 In mice, only a single type of trypsin has been identified.9
Past studies have shown that trypsins or trypsin-like enzymes are produced by human cancer cells of the stomach,10,11 ovary,12 lung,13 colon,14 and others.14 The tumor-derived trypsin is likely to contribute to tumor invasion and metastasis by degrading extracellular matrix proteins and by activating the latent forms of matrix metalloproteinases (MMPs).10-12 Recently, we found that trypsin 2 is expressed by vascular endothelial cells around gastric tumors and in patients with disseminated intravascular coagulation.15 Trypsin-like enzymes have been observed in some normal tissues.13,16,17 The trypsin 4 gene is expressed in the human brain.7 These facts suggest broad distribution and function of trypsin in normal tissues. In the present study, we surveyed the expression of trypsin mRNA and protein in various human and mouse tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All human tissues were obtained by autopsy performed less than 3 hours postmortem and were immediately fixed in 10% formalin. The paraffin-embedded sections were mounted on aminoacyl silane-coated glass slides and used for in situ hybridization and immunohistochemical analysis. Normal mouse tissues were obtained from 8-week-old female ICR mice, washed with cold phosphate-buffered saline (PBS), and stored at -40°C until use. Digestive tract tissues were washed with PBS containing 0.1% (w/v) sodium dodecyl sulfate to avoid contamination of pancreatic trypsin and then washed further with PBS alone. The frozen tissues were thawed, homogenized in 5 volumes (w/v) of 50 mmol/L Tris-HCl (pH 7.5) containing 0.1% (w/v) Triton X-100, and cleared by centrifugation. For activation of trypsinogen, 10 µl of the tissue extract (5 mg protein/ml) was mixed with 2 µl of 50 µg/ml enterokinase (Biozyme; South Wales, UK) and 3 µl of 50 mmol/L CaCl2 and incubated at 37 °C for 1 hour, followed by mixing with an equal volume of 2x sodium dodecyl sulfate sample buffer without 2-mercaptoethanol.
Northern Blotting and in Situ Hybridization
Nylon membranes blotted with polyadenylated RNAs, Multiple Tissue Northern Blots (Clontech Laboratories, Palo Alto, CA), were used to analyze expression of trypsin mRNAs in various human tissues except for the skin. Polyadenylated RNA from human skin was purchased from Nippon Gene (Tokyo, Japan) and used for Northern blotting analysis of trypsin mRNAs. Hybridization of trypsin mRNAs with a 32P-labeled trypsin 1 cDNA (nucleotides 1 to 482 in Ref. 5 ) and subsequent autoradiography were carried out as described before.12,15 For in situ hybridization analysis of trypsin messages in various human tissues, sense and antisense RNA probes were prepared from a cDNA fragment (nucleotides 131 to 482 in Ref. 5 ) of human trypsin 1.15 In situ hybridization was carried out as reported previously.15 The hybridized signals were visualized by the alkaline phosphatase reaction. In this analysis, the RNA probe was expected to hybridize to trypsin 1, 2, 3, and 4 mRNAs under the experimental conditions used. The tissue sections were counterstained with methyl green.
Reverse Transcription-Polymerase Chain Reaction and Nucleotide Sequences
For reverse transcription-polymerase chain reaction (RT-PCR) analysis, the following two primers were designed: sense, 5'-CTACAAATACCGCATCCAAGT-3', and antisense, 5'-ACCTCGTCCAGACCCAACAA-3', which corresponded to nucleotides 208 to 228 and 473 to 492, respectively, of mouse trypsin (or trypsinogen) cDNA.9 For nucleotide sequencing, RT-PCR products of approximately 280 bp were electrophoretically separated and cloned into a pGEM-T Easy vector (Promega, Madison, WI). The nucleotide sequences of the cloned DNA fragments were determined by the dideoxynucleotide method, using cycle sequencing with a LI-COR automatic sequencer (Lincoln, NE).
Immunohistochemistry
Immunohistochemical staining of human tissues for trypsin was carried out with a mouse monoclonal antibody raised against human pancreatic trypsin (Chemicon; Temecula, CA), as described before.12 Briefly, 4-µm-thick paraffin sections were dewaxed, rehydrated, and immersed in 0.3% hydrogen peroxide-containing methanol for inactivation of intrinsic peroxidase. Then the sections were incubated with the anti-trypsin monoclonal antibody at 37°C for 1 hour. The labeled antigen was visualized by the 3,3'-diaminobenzidine reaction with a HistoFine kit (Nichirei, Tokyo, Japan). The trypsin antibody was reactive to pancreatic trypsins/trypsinogens 1 and 2 as tested by immunoblotting.
Gelatin Zymography
Unless otherwise noted, gelatin zymography was carried out on 14% polyacrylamide slab gels (90 mm long, 90 mm wide, and 0.75 mm thick) under nonreducing conditions, as described before.10,15 Molecular weight markers were purchased from Bio-Rad (Richmond, CA). To distinguish metalloproteinases and serine proteinases, enterokinase-treated tissue extracts were incubated with 5 mmol/L ethylenediaminetetraacetic acid or with 10 mmol/L diisopropyl fluorophosphate.
Determination of Protein Concentration
Protein concentration was determined by the dye method with a Bio-Rad protein assay kit (Bio-Rad), using bovine immunoglobulin G as the standard.
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Results |
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Expression of trypsin in various human normal tissues was analyzed
by Northern blotting with trypsin 1 cDNA as a probe, which presumably
hybridizes to trypsin 2, 3, and 4 mRNAs, as well as trypsin 1 mRNA. As
shown in Figure 1
, trypsin mRNA of
approximately 0.85 kb was detected strongly in the pancreas and spleen
and clearly in the small intestine. Other tissues, including the T
lymphocyte-producing tissue thymus, did not show the hybridization
signal for the transcript.
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When the spleen was subjected to in situ hybridization, the
cells in the white pulp showed a strong signal for trypsin mRNA (Figure 2A)
. The hybridization signal was
especially strong in larger cells, which appeared to be macrophages and
monocytes. Many lymphocytes in the white pulp also showed a positive
signal for trypsin mRNA. In contrast, the trypsin message was faint, if
present at all, in spleen cells of the red pulp (data not shown).
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. Stratified squamous epithelial cells expressed
trypsin message in the esophagus and skin. In the esophagus, the signal
for trypsin mRNA was more intense in the basal and parabasal cells
(Figure 2D)
, whereas in the skin, the signal was evenly distributed
from the basal cells to the stratum spinous cells (Figure 2B)
. In the
kidney, the epithelial cells of the collecting ducts were densely
stained with the antisense probe, and those of other segments of
nephron were weakly stained (Figure 2E)
. In the lung, the bronchial and
bronchiolar epithelial cells were focally stained for trypsin
transcript (Figure 2G)
. In the liver, hepatocytes clearly showed the
positive signal for trypsin mRNA (Figure 2F)
. Gastric epithelial cells
were also positive for trypsin message, but the thyroid, testis, and
cardiac muscle did not show specific reactivity to the trypsin
antisense probe (data not shown).
To verify the synthesis of trypsin in the human tissues,
immunohistochemical analysis with the anti-trypsin antibody was carried
out (Figure 3)
. The immunoreactivity to
trypsin showed essentially the same distribution as that of the trypsin
message detected by in situ hybridization in all of the
tissues except the skin. In the skin, the trypsin antigen was more
abundantly detected in the basal keratinocytes than in the stratum
spinosum (Figure 3A)
.
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. Both
trypsin message and protein were detected in nerve cells of the
hippocampus and the cerebral cortex.
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To confirm the presence of tryptic activity in nonpancreatic
tissues, various tissues of normal mice were extracted and analyzed
by gelatin zymography in the absence of Ca2+ before and
after activation by enterokinase (Figure 5)
. When pretreated with enterokinase,
the pancreas showed a high gelatinolytic activity at 23 kd, which
appeared to correspond to the active form of trypsin (Figure 5A)
. The
23-kd gelatinolytic activity was strongly inhibited when the extract
was treated with the serine proteinase inhibitor diisopropyl
fluorophosphate (data not shown). Furthermore, immunoblotting analysis
with a polyclonal antibody against human pancreatic trypsin confirmed
that the 23-kd gelatinolytic activity was due to mouse trypsin (data
not shown).
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. The trypsin activity was high in
the pancreas, skin, small intestine, esophagus, and stomach and
moderate in the brain, spleen, and kidney. Even in the absence of
enterokinase, almost the same level of trypsin activity was detected in
the skin and small intestine. In the esophagus, stomach, and large
intestine, the trypsin activity was detected more weakly in the absence
of enterokinase than in its presence. These results indicated that
these tissues contained an endogenous activator of trypsinogen, most
likely enterokinase. The remaining tissues showed little activity at 23
kd in the absence of enterokinase treatment, suggesting that they
contained a very low level, if any, of enterokinase.
The gelatin zymography also exhibited gelatinolytic activities with
approximate molecular sizes of 100, 70, and 40 kd, in addition to the
23-kd activity (Figure 5)
. These activities were especially high in the
enterokinase-treated extracts of the spleen, brain, esophagus, stomach,
and large intestine. In other experiments with trypsin-secreting human
cancer cell lines, we have found that similar gelatinolytic activities
are produced by sodium dodecyl sulfate-stable complexes of active
trypsin with secreted forms of Alzheimer amyloid protein precursor (or
protease nexin II) and their partially degraded products (unpublished
data). Alzheimer amyloid protein precursors, which contain the
Kuniz-type serine proteinase inhibitor domain, are known to be secreted
from various types of cultured cells.18
Therefore, it is
most likely that the gelatinolytic activities at 100, 70, and 40 kd in
the tissue extracts are derived from the trypsin-Alzheimer amyloid
protein precursor complexes.
PCR Analysis of Trypsin Genes Expressed in Normal Mouse Tissues
To determine trypsin species expressed in some normal tissues of mice, we carried out RT-PCR analysis for trypsin mRNAs and subsequent sequencing of the amplified DNA fragments. The RT-PCR amplified a 280-bp product from the RNAs isolated from the brain, kidney, spleen, and liver. Each DNA product was isolated, cloned into a plasmid vector, and sequenced. The sequence analyses showed that the nucleotide sequences of the amplified 280-bp products from the brain, kidney, and spleen were completely identical to that of mouse trypsin cDNA (nucleotides 208 to 492), indicating that at least these tissues express the pancreatic trypsin gene (data not shown). The sequence of the PCR product from the liver RNA was very similar but not identical to that of the trypsin cDNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unexpectedly, the spleen was the highest in trypsin expression of all of the tissues except for the pancreas. Monocytes and macrophages in the white pulp of spleen showed a strong signal for trypsin transcript, whereas lymphocytes showed a weak signal. It is known that many types of leukocytes constitutively secrete gelatinase B (MMP-9) and neutrophil collagenase (MMP-8), which are effectively activated by trypsin.22,23 Activated human B cells and B-cell lines produce a trypsin-like proteinase,24 whereas T lymphocytes produce several types of granzymes25 and tryptase TL2.26 Very recently, Fukusen and Aoki17 purified two forms of novel serine proteinases with high similarity to trypsin from mouse spleen. Our PCR analysis showed that mouse spleen expressed the pancreatic trypsin gene at a high level. It is speculated that the splenic trypsin plays some roles in the immune defense.
The small intestine and stomach expressed trypsin at a high level. Jeohn et al16 purified a trypsin from the intracellular microsomal fraction of porcine gastric mucosa. We have previously found that gastric adenocarcinoma cells secrete trypsins 1 and 2 in latent and active forms.11,27 Therefore, we assume that in the stomach and intestine, trypsins are secreted and function as digestive enzymes together with the pancreas-derived trypsins. Similarly, trypsin produced by various types of epithelial cells, especially of exocrine glands such as the bile duct and the nephron of the kidney, may function in the digestion of unnecessary excreted proteins as a "pipe cleaner."
The stratified squamous epithelia in the esophagus and skin expressed trypsin at relatively high levels. In mice, the extracts from these epithelia, as well as the stomach and intestine, contained the active form of trypsin. This implies that in these tissues the secreted latent trypsin (trypsinogen) is continuously activated to the active trypsin by an endogenous activator, probably enterokinase. Recently Yahagi et al28 have reported that enterokinase gene is expressed in the digestive tract. Proteolytic degradation of intercellular cohesive structures in the stratum corneum is required for desquamation, ie, the cell shedding at the skin surface.29 Recently, a skin-specific novel serine proteinase called stratum corneum chymotryptic enzyme has been characterized and is expected to be involved in desquamation.30 Stratum corneum chymotryptic enzyme has homology to trypsin, chymotrypsin, and kallikrein, and its proenzyme is effectively activated by trypsin. It seems likely that the enterokinase-activated trypsin is involved in the desquamation both by directly degrading the intercellular cohesive structures and by activating the latent forms of stratum corneum chymotryptic enzyme and MMPs. As another possibility, trypsin may be involved in the growth and terminal differentiation of epidermal keratinocytes.
The present study also demonstrated the expression of trypsin gene in human and mouse brains. Trypsin 4 cDNA, the protein product of which does not have a signal sequence, has been cloned from human brain cDNA library.7 In addition, cDNAs for two trypsin-like enzymes expressed in the human brain, neuropsin31 and neurosin,32 have been cloned. Neuropsin is specifically expressed in pyramidal neurons of the hippocampus. The physiological roles of these trypsins and trypsin-related enzymes in the central nervous system seem to be among the most important subjects to be studied.
Ubiquitus expression of trypsin genes suggests their essential roles in the maintenance of various cellular functions. Furthermore, aberrant expression of tissue trypsin is likely to be involved in various pathological processes, including tumor invasion and inflammation. Further studies are required for clarifying the detailed functions of trypsin in each type of tissue and cell.
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Footnotes |
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Supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (to NK) and Grants-in-Aid from the Uehara Memorial Foundation (to KMiy), from the Ministry of Health and Welfare (to KMiy), and from the Ministry of Education, Culture, Sports and Science of Japan (to YN and KMiy).
Accepted for publication June 26, 1998.
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