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Human urinary trypsin inhibitor (urinastatin)-like substance in mouse liver
Life Sciences, Vol. 50, pp. 1399-1406 Printed in the USA
HUMAN URINARY TRYPSIN
Pergamon Press
INHIBITOR (URINASTATIN)-LIKE SUBSTANCE MOUSE LIVER
IN
Tadahiro S h i k i m i 1 ' * , Tong H. J o h 3,
S y u s a k u S u z u k i 2, Thomas Wessel 3, K e i s u k e H a t t o r i 1 and S h u j i T a k a o r i 1
1Department of Pharmacology and 21nstitute of Experimental Animals, 3 Shimane Medical University, Izumo, Shimane 693, Japan and Laboratory of Molecular Neurobiology, The W. M. Burke Medical Research Institute, Cornell University Medical College, White Plains, New York 10605, U.S.A. (Received in final form February 25, 1992) Summary Mouse liver contains a human urinary trypsin inhibitor (urinastatin, UT)-like immunoreactive substance with trypsin inhibitory activity. N o r t h e r n blot analysis demonstrates the presence of the appropriate 1.3 kb mRNA band in liver tissue but not in kidney or other tissues examined. A d m i n i s t r a t i o n of hydrocortisone, which is known to increase the urinary excretion of the UT-like substance, increased the levels of UT-like substance in serum and in the liver tissue. In contrast, d e o x y c o r t i c o s t e r o n e acetate did not have such an effect. These results suggest that the gene encoding UT-like substance is primarily expressed in the liver of the mouse, and that glucocorticoids play an important role in regulating the hepatic synthesis of UT-like substance. Furthermore, these findings indicate that the mouse is a suitable species for research on the biological function of UT or UT-like substances. Urinastatin (UT) is a trypsin inhibitor which is found in human urine. UT is normally excreted at the level of several mg per liter in human urine, however, little is known about its biological function. We reported previously that a UT-like immunoreactive substance with trypsin inhibitory activity is found in mouse urine, but is not detectable in the urine of other animal species examined (i). This paper extends these observations to examine the site of synthesis, the biochemical properties with respect to trypsin inhibitory function, the effect of glucocorticoids, and the d i s t r i b u t i o n of the mRNA for UT in the mouse. Our results indicate that the mouse is indeed a suitable species to examine UT gene e x p r e s s i o n and regulation, and for research on the biological function of UT or UT-like substances. * TO w h o m c o r r e s p o n d e n c e should be addressed. 0024-3205/92 $5.00 + .00 Copyright o 1992 Pergamon Press Ltd All rights reserved.
1400
Urinastatin-like Substance in Mouse Liver
Materials
Vol. 50, No. 19, 1992
and Methods
Materials: Urinastatin (UT) was kindly provided by Mochida Pharmaceutical Co. Japan. This purified protein migrates as a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and in Sephadex gel chromatography. The following reagents were used: CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Sweden); gelatin (Difco Laboratories, U.S.A.); trypsin (Miles Laboratories, U.S.A.); Dako peroxidase-antiperoxidase kit (Dako Corporation, U.S.A.); deoxycorticosterone acetate (DOCA) (Wako Pure Chemical Industries, Japan); hydrocortisone sodium phosphate (Banyu Pharmaceutical Co., Japan); guanidinium thiocyanate (Fluka Chemie AG, Switzerland); RNA molecular weight marker #i (0.3 - 7.4 kb), and oligo(dT) cellulose (Boehringer Mannheim Biochemicals, U.S.A.); Gene Screen Plus nylon membrane (Dupont NEN, U.S.A.). Animals: used.
Male
ICR mice
(Clea,
Japan)
weighing
20-25
g were
Anti-UT sera: Anti-UT sera were raised in rabbits as described in a previous report (i). The complement activity in the sera was destroyed by heating at 56°C for 30 min. The IgG fraction of anti-UT sera was obtained by fractionation with Na2SO 4 followed by passage through a DEAE-cellulose column. Tissue extract and serum preparation: Mice were anesthetized with pentobarbi£al sodium (40 mg/kg, i.p.) and perfused through the aorta with 0.9 % NaCI. Kidney and liver tissues were removed, weighed and homogenized on ice in a Potter Elvehjem glass homogenizer with 10 volumes of distilled water. The homogenate was centrifuged at 13,000 x g for 10 min at 4°C and the supernatant was used as the tissue extract. Blood was collected from left ventricle of the heart before perfusion and was kept at 4°C for 20 hrs. Serum was separated by centrifugation. Affinity chromatography of the tissue extract: Kidney or liver extracts, equivalent to 8 g of wet weight of tissue, or 1 ml of serum were lyophilized and subseqently dissolved in 4 ml of Tris-HCl buffer (4 mM, pH 8.2) containing 0.5 M NaCI. These samples were then applied to a CNBr-activated Sepharose 4B column (Ig, bed volume 2.4 ml) which had previously been coupled with 15 mg of rabbit anti-UT IgG by the method described in the Pharmacia manual. The equilibration buffer was the same as used to dissolve the samples (Tris-HCl 4 mM, pH 8.2 in 0.5 M NaCI). Acetic acid (4 mM, pH 3.6) in 0.5 M NaCI served as the elution buffer. The eluate was dialysed against distilled water and then lyophilized. Assay of UT-like immunoreactivit~ and trypsin inhibition: The level of UT-like substance was measured by using a sandwich enzyme immunoassay (i). Trypsin inhibitory activity was detected by using gelatin-containing SDS-PAGE, according to the method described by Hanspal et al. (2): The method involved (a) incorporation of gelatin into the SDS-polyacrylamide gel at the time of casting; (b) elecrophoresis of the sample (which was prepared in 2.5 % SDS without heating) in the presence of SDS; (c) removal of SDS by washing the gel in Triton X-100; (d) incubation of the gel in a solution containing trypsin; and (e) detection of trypsin inhibitory activity by staining for undigested gelatin.
Vol. 50, No. 19, 1 9 9 2
Urinastatin-like Substance in Mouse Liver
1401
A d m i n i s t r a t i o n of dru~s: Bilaterally adrenalectomized mice were m a i n t a i n e d on 0.9 % physiological saline as the sole source of drinking fluid. On the seventh p o s t o p e r a t i v e day, hydrocortisone (10 mg/kg, i.v.) and solvent (0.9 % physiological saline); d e o x y c o r t i c o s t e r o n e acetate (50 mg/kg, s.c.) and vehicle (sesame oil) were injected between 9:00 and i0:00 A.M.. T w e n t y - f o u r hours later, liver and kidney extracts and serum were prepared as described in the above section. Immunohistochemistr[: Animal were sacrificed by carotid exsanguination. Liver and kidney were removed and fixed in Zamboni solution (3), embedded in paraffin and the tissue was cut at 6 2am thickness. After removal of the paraffin, the d i s t r i b u t i o n of UT-like substance in the tissue was examined by using the polyclonal anti-UT serum described above. The secondary antibody and chromogen reaction were done according to the protocol outlined in the Dako peroxidase-anti- peroxidase kit. N o r t h e r n blot: Total RNA was prepared using the g u a n i d i n i u m t h i o c y a n a t e - p h g n o l - c h l o r o f o r m procedure of Chomczynski and Sacchi (4). Poly(A)'mRNAS were subsequently extracted by the +use of oligo(dT) cellulose as described by Kingston (5). Poly(A) mRNAs were analysed by Northern blot as described elsewhere in detail (6). The CDNA probe for human UT is a 1 . 2 ~ b EcoRI fragment from human liver cDNA and was labelled with (J~P)dCTP by random priming (Random primed labelling kit; Boehringer Mannheim Biochemicals, U.S.A.). Results Mouse serum and extracts of mouse liver and kidney contain multiple substances of various molecular weight that inhibit trypsin activity (Fig.iA). Serum as well as liver and kidney extracts eluated of the anti-UT-IgG affinity column show inhibitory bands corresponding in size to that of purified UT, as depicted in Fig.lB.. H y d r o c o r t i s o n e and DOCA have different effects on the tissue and the serum levels of UT-like immunoreactive substance in the a d r e n a l e c t o m i z e d mouse. Hydrocortisone increased the liver and the serum levels of UT-like immunoreactive substance, while the renal levels did not change significantly. DOCA had no effect on tissue or serum levels of UT-like substance (Fig. 2). In the liver tissue, UT-like immunoreactivity was observed diffusely in the parenchyma, in contrast, in the kidney immunoreactivity was localized to the distal tubules, but not observed in the glomeruli, proximal tubules or the papillary ducts. A striking increase in immunoreactivity was observed in the liver of hydrocortisonetreated mice when compared to saline-injected mice, while no differences in renal staining could be detected in these two groups (data not shown). Northern blot analysis provides proof of specific h y b r i d i z a t i o n of the human UTcDNA probe to a single 1.3 kb transcript in the mouse liver which corresponds exactly to the mRNA size in human tissue (Fig.3). No signal could be detected with this cDNA probe in murine kidney, heart and lung.
1402
Urinastatin-like Substance in Mouse Liver
Vol. 50, No. 19, 1992
Discussion In humans, UT or UT-like substance is believed to be derived from human serum inter- ~ - t r y p s i n inhibitor (ITI), which is synthesized in liver tissue (7-9). S c h r e i t m 6 1 1 e r et al. (8,10) have suggested that ITI is a tight complex of three subunits. The smallest subunit in this large molecule is UT. In contrast, Salier et al. (9,11) contend that ITI consists of covalently linked light and heavy chains, and that UT is part of the light chain. In the present study, we have found that mouse liver possesses a protein with trypsin inhibitory properties that coincides in m o l e c u l a r w e i g h t with that of human UT. Furthermore, a 1.3 kb mRNA species was identified in mouse liver with a cDNA probe specific to human UT. This matches the size of the human UT
A 1
2
B 3
4
1
FIG.
2
3
1
Trypsin inhibitory activity demonstrated by using gelatin containing SDS-PAGE. A: lane i, UT (40 ng); lane 2, mouse liver extract equivalent to 40 mg of original wet weight; lane 3, mouse serum (0.5 ~i); lane 4, mouse kidney extract equivalent to 40 mg of original wet weight. B: lane I, UT (40 ng); lane 2, eluate of mouse liver extract from the anti-UT-IgG affinity column; lane 3, eluate of mouse serum from the a n t i - U T - I g G affinity column; lane 4, eluate of mouse kidney extract from the anti-UT-IgG affinity column.
4
vol. 50, No. 19, 1 9 9 2
Urinastatin-like Substance in Mouse Liver
1403
transcript which encodes a 40 kDa polypeptide chain found in human liver (9). This result supports the concept that ITI with its molecular w e i g h t of 150-160 kDa is in fact not a single chain protein but rather that UT is an integral component of ITI. In addition, the UT-like immunoreactive substance with trypsin inhibitory activity in the mouse liver, kidney and serum was found to have a nearly same molecular weight as that of UT. UT and UT-like substances are thought to be released by d i s s o c i a t i o n of the subunits of ITI w h i c h may possibly involve limited proteolysis of ITI. In vitro studies have shown that UT is released from ITI by proteolytic enzymes, such as granulocyte elastase (12), trypsin (12,13), chymotrypsin ( 1 3 ) and kallikrein (14) as well as by acidic conditions (i0).
12
O.IE
0.9
l
°l---
~ ._ 0.8
Liver
~._.
Serum
Kidney i.
o
o-~
~-
f
c97
= "~- 0.7
.!
¢_ ~
11
i
°w--
0.~
' i~'~0.05 ~4.a
e--
~.~
¢1~
0
-,~
9
I
0
0 5O HC DOCA (mg/kg)
0
HC
DOCA
(mg/kg)
FIG.
~
5 5 51 5 O" 0 lO 0 50 HC DOCA (mg/kg)
2
Changes in the levels of urinastatin-like substance in the adrenalectomized mice. Twenty-four hours after the a d m i n i s t r a t i o n of the drugs or vehicle, serum and tissue extracts of liver and kidney were prepared. UTlike immunoreactive substance was determined by the sandwich enzyme immunoassay. Numbers of d e t e r m i n a t i o n are shown in parenthesis. Statistical analysis were performed by Student's t-test. *P<0.05 HC: h y d r o c o r t i s o n e DOCA: d e o x y c o r t i c o s t e r o n e acetate
1404
Urinastatin-like Substance in Mouse Liver
Vol. 50, No. 19, 1992
In humans, the a d m i n i s t r a t i o n of g l u c o c o r t i c o i d s enhances the e x c r e t i o n of UT (15). This finding was r e p l i c a t e d in mice (i): adrenalectomy decreased urinary excretion of the UT-like immunoreactive substance, and administration of ACTH or hydrocortisone increased the excretion of this substance. Furthermore, the mineralocorticoid DOCA did not produce the same effect (i). In the present study, hydrocortisone increased urinary e x c r e t i o n of UT-like substance as well as liver and the serum levels of this protein, whereas DOCA did not. Mouse kidney tissue also contains a UT-like i m m u n o r e a c t i v e substance (16), and its level in h y d r o c o r t i s o n e - u n t r e a t e d mice is about o n e - f i f t h of that in the liver tissue. The level of i m m u n o r e a c t i v i t y in the kidney tissue did not appear to be affected by the a d m i n i s t r a t i o n of hydrocortisone. Using equal amounts of p o l y ( A ) - R N A s from several tissues, a single m R N A band of the a p p r o p r i a t e size could
kb 7.4-5.3-FIG.
2.8-1.91.64 - 1.3 kb 1.00.60.40.3-
3
N o r t h e r n blot analysis of mouse liver RNA. Poly(A) + RNAs in the liver tissue was prepared, and 1.2 ~ g of mRNA sample was loaded on a 1 % agarose gel containing 6 % formaldehyde, and run at 1 V/cm for 16 hr. Following capillary transfer of mRNAs to a Gene Screen Plus nylon membrane, the filter was probed with a random-primed human UT probe and exposed to film for 2 days. Size of the UTmRNA was calculated with RNA molecular weight markers shown in left side.
Vol. 50, No. 19, 1 9 9 2
Urinastatin-like Substance in Mouse Liver
1405
be clearly d e m o n s t r a t e d for the liver with the human UTcDNA probe, while no such band could be detected for the renal tissue. This observation is reminiscent of a finding by Salier and his associates in baboon tissues and human cell lines that the mRNA for the light chain of ITI (which these investigators believe to be a UT precursor) is found only in liver and not in other tissues, such as the kidney (17). The fact that pronounced increases in UT-like immunoreactivity could be detected in the liver and serum, while renal staining was virtually unchanged despite enhanced urinary excreation of this protein lends further support to the concept that the liver is the primary and probably sole site of UT synthesis. It is not clear whether ITI is actively dissembled in the distal tubule cells or whether dissociated UT is somehow taken up and possibly modified before excretion into the urine. Overall, the biological function of UT remains quite puzzling: a tumor-associated trypsin inhibitor which is identical to UT acts as a growth factor for endothelial cells in culture (18); the precursor of amyloid B-protein in Alzheimer's disease, has an amino acid sequence which is nearly 50 % similar to the inhibitory domain of UT (19), and trypsin bound to UT readily dissociates in the presence of e~-proteinase inhibitor or e2-macroglobulin to form a more ~table complex with this antiproteinases than does UT (20). We previously reported that amongst urine from dogs, cats, rabbits, guinea-pigs, rats and mice, only mouse urine contains a UT-like immunoreactive substance with trypsin inhibitory activity (i). The present experiments indicate that mouse liver contains a gene transcript that shows specific hybridization with a human UT probe and has a comparable size on N o r t h e r n blot. Whether the UT-like substance in mouse tissue is identical or highly homologus to the human UT requires further study, however, the present finding is convincing evidence that mouse is a suitable species for research on the biological functions of UT or UT-like substances. Acknowledgements The cDNA probe for human UT was generously provided by D r . W . G e b h a r d and Prof. K.Hochstrasser of the Ludwig-Maximilians University, M~nich, Germany. References i. T. SHIKIMI, S. SUZUKI, M. TAKAHASHI and H. KANETO, Scand. J. Clin. Lab. Invest. 50 1-8 (1990). 2. J.S. HANSPAL, G.R. B-~SHELL and P. GHOSH, Anal. Biochem. 132 288-293 (1983). 3. M. STEFANINI, C. DE MARTINO and L. ZAMBONI, Nature (London) 216 173-174 (1967). 4. P. CHOMCZYNSKI and N. SACCHI, Anal. Biochem. 162 156-159 (1987). 5. R.E. KINGSTON, Current Protocols in M o l e c u l a r Biology, F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl (eds) Vol.2, pp. 4.5.1-4.5.3., Greene P u b l i s h i n g Associates and Wiley Interscience, New York (1987). 6. D.M. STONE, T. WESSEL, T.H. JOH and H. BAKER, Mol. Brain Res.
1406
Urinastatin-like Substance in Mouse Liver
Vol. 50, No. 19, 1992
8 291-300 (1990). 7. J.F. KAUMEYER, J.O. POLAZZI and M.P. KOTICK, Nucl. Acids. Res. 14 7839-7850 (1986). 8. T. SC~-REITMULLER, K. HOCHSTRASSER, P.W.M. REISINGER, E . W A C H T E R and W. GEBHARD, Biol. Chem. H o p p e - S e y l e r 368 963-970 (1987). 9. J. BOURGUIGNON, M. DIARRA-MEHRPOUR, R. SESBOUE, M. FRAIN, J.M. SALA-TREPAT, J.P. MARTIN and J.P. SALIER, Biochem. Biophys. Res. Comm. 131 1146-1153 (1985). 10. W. GEBHARD, G. LEYSATH and T. SCHREITMULLER, Biol. Chem. H o p p e - S e y e r 369 Suppl. 19-22 (1988). ii. J-P. SALIER, M. DIARRA-MEHRPOUR, R. SESBOUE, J. BOURGUIGNON and J-P. MARTIN, Biol. Chem. Hoppe-Seyer 369 Suppl. 15-18 (1988). 12. T. DIETL, W. DOBRINSKI and K. HOCHSTRASSER, Hoppe-Seyler's Z. Physiol.Chem. 360 1313-1318 (1979). 13. P. REISINGER, K. HOCHSTRASSER, G.J. ALBRECHT, K. LEMPART and J-P. SALIER, Biol. Chem. Hoppe-Seyler 366 479-483 (1985). 14. G. BRETZEL and K. HOCHSTRASSER, Hoppe-Seyler's Z. Physiol. Chem. 357 487-489 (1976). 15. H.J. FAARVANG, Acta Pharmacol. Toxicol. 19 293-304 (1962). 16. T. SHIKIMI and S. SUZUKI, Biol. Chem. Hoppe-Seyer 371 991-997 (1990). 17. J.P. SALIER, M. DIARRA-MEHRPOUR, R. SESBOUE, J. BOURGUIGNON, R. BENAROUS, I. OHKUBO, S. KURAUCHI, K. KURAUCHI and J.P. MARTIN, Proc. Natl. Acad. Sci. USA 84 8272-8276 (1987). 18. W.L. MCKEEHAN, Y. SAKAGAMI, H. HOSHI and K.A. MCKEEHAN, J. Biol. Chem. 261 5378-5383 (1986). 19. R.W. CARRELL, Nature (London) 331 478-479 (1988). 20. B-H. JONSSON and K. OHLSSON, H o p p e - S e y l e r ' s Z. Physiol. Chem. 365 1403-1408 (1984).
HUMAN URINARY TRYPSIN
Pergamon Press
INHIBITOR (URINASTATIN)-LIKE SUBSTANCE MOUSE LIVER
IN
Tadahiro S h i k i m i 1 ' * , Tong H. J o h 3,
S y u s a k u S u z u k i 2, Thomas Wessel 3, K e i s u k e H a t t o r i 1 and S h u j i T a k a o r i 1
1Department of Pharmacology and 21nstitute of Experimental Animals, 3 Shimane Medical University, Izumo, Shimane 693, Japan and Laboratory of Molecular Neurobiology, The W. M. Burke Medical Research Institute, Cornell University Medical College, White Plains, New York 10605, U.S.A. (Received in final form February 25, 1992) Summary Mouse liver contains a human urinary trypsin inhibitor (urinastatin, UT)-like immunoreactive substance with trypsin inhibitory activity. N o r t h e r n blot analysis demonstrates the presence of the appropriate 1.3 kb mRNA band in liver tissue but not in kidney or other tissues examined. A d m i n i s t r a t i o n of hydrocortisone, which is known to increase the urinary excretion of the UT-like substance, increased the levels of UT-like substance in serum and in the liver tissue. In contrast, d e o x y c o r t i c o s t e r o n e acetate did not have such an effect. These results suggest that the gene encoding UT-like substance is primarily expressed in the liver of the mouse, and that glucocorticoids play an important role in regulating the hepatic synthesis of UT-like substance. Furthermore, these findings indicate that the mouse is a suitable species for research on the biological function of UT or UT-like substances. Urinastatin (UT) is a trypsin inhibitor which is found in human urine. UT is normally excreted at the level of several mg per liter in human urine, however, little is known about its biological function. We reported previously that a UT-like immunoreactive substance with trypsin inhibitory activity is found in mouse urine, but is not detectable in the urine of other animal species examined (i). This paper extends these observations to examine the site of synthesis, the biochemical properties with respect to trypsin inhibitory function, the effect of glucocorticoids, and the d i s t r i b u t i o n of the mRNA for UT in the mouse. Our results indicate that the mouse is indeed a suitable species to examine UT gene e x p r e s s i o n and regulation, and for research on the biological function of UT or UT-like substances. * TO w h o m c o r r e s p o n d e n c e should be addressed. 0024-3205/92 $5.00 + .00 Copyright o 1992 Pergamon Press Ltd All rights reserved.
1400
Urinastatin-like Substance in Mouse Liver
Materials
Vol. 50, No. 19, 1992
and Methods
Materials: Urinastatin (UT) was kindly provided by Mochida Pharmaceutical Co. Japan. This purified protein migrates as a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and in Sephadex gel chromatography. The following reagents were used: CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Sweden); gelatin (Difco Laboratories, U.S.A.); trypsin (Miles Laboratories, U.S.A.); Dako peroxidase-antiperoxidase kit (Dako Corporation, U.S.A.); deoxycorticosterone acetate (DOCA) (Wako Pure Chemical Industries, Japan); hydrocortisone sodium phosphate (Banyu Pharmaceutical Co., Japan); guanidinium thiocyanate (Fluka Chemie AG, Switzerland); RNA molecular weight marker #i (0.3 - 7.4 kb), and oligo(dT) cellulose (Boehringer Mannheim Biochemicals, U.S.A.); Gene Screen Plus nylon membrane (Dupont NEN, U.S.A.). Animals: used.
Male
ICR mice
(Clea,
Japan)
weighing
20-25
g were
Anti-UT sera: Anti-UT sera were raised in rabbits as described in a previous report (i). The complement activity in the sera was destroyed by heating at 56°C for 30 min. The IgG fraction of anti-UT sera was obtained by fractionation with Na2SO 4 followed by passage through a DEAE-cellulose column. Tissue extract and serum preparation: Mice were anesthetized with pentobarbi£al sodium (40 mg/kg, i.p.) and perfused through the aorta with 0.9 % NaCI. Kidney and liver tissues were removed, weighed and homogenized on ice in a Potter Elvehjem glass homogenizer with 10 volumes of distilled water. The homogenate was centrifuged at 13,000 x g for 10 min at 4°C and the supernatant was used as the tissue extract. Blood was collected from left ventricle of the heart before perfusion and was kept at 4°C for 20 hrs. Serum was separated by centrifugation. Affinity chromatography of the tissue extract: Kidney or liver extracts, equivalent to 8 g of wet weight of tissue, or 1 ml of serum were lyophilized and subseqently dissolved in 4 ml of Tris-HCl buffer (4 mM, pH 8.2) containing 0.5 M NaCI. These samples were then applied to a CNBr-activated Sepharose 4B column (Ig, bed volume 2.4 ml) which had previously been coupled with 15 mg of rabbit anti-UT IgG by the method described in the Pharmacia manual. The equilibration buffer was the same as used to dissolve the samples (Tris-HCl 4 mM, pH 8.2 in 0.5 M NaCI). Acetic acid (4 mM, pH 3.6) in 0.5 M NaCI served as the elution buffer. The eluate was dialysed against distilled water and then lyophilized. Assay of UT-like immunoreactivit~ and trypsin inhibition: The level of UT-like substance was measured by using a sandwich enzyme immunoassay (i). Trypsin inhibitory activity was detected by using gelatin-containing SDS-PAGE, according to the method described by Hanspal et al. (2): The method involved (a) incorporation of gelatin into the SDS-polyacrylamide gel at the time of casting; (b) elecrophoresis of the sample (which was prepared in 2.5 % SDS without heating) in the presence of SDS; (c) removal of SDS by washing the gel in Triton X-100; (d) incubation of the gel in a solution containing trypsin; and (e) detection of trypsin inhibitory activity by staining for undigested gelatin.
Vol. 50, No. 19, 1 9 9 2
Urinastatin-like Substance in Mouse Liver
1401
A d m i n i s t r a t i o n of dru~s: Bilaterally adrenalectomized mice were m a i n t a i n e d on 0.9 % physiological saline as the sole source of drinking fluid. On the seventh p o s t o p e r a t i v e day, hydrocortisone (10 mg/kg, i.v.) and solvent (0.9 % physiological saline); d e o x y c o r t i c o s t e r o n e acetate (50 mg/kg, s.c.) and vehicle (sesame oil) were injected between 9:00 and i0:00 A.M.. T w e n t y - f o u r hours later, liver and kidney extracts and serum were prepared as described in the above section. Immunohistochemistr[: Animal were sacrificed by carotid exsanguination. Liver and kidney were removed and fixed in Zamboni solution (3), embedded in paraffin and the tissue was cut at 6 2am thickness. After removal of the paraffin, the d i s t r i b u t i o n of UT-like substance in the tissue was examined by using the polyclonal anti-UT serum described above. The secondary antibody and chromogen reaction were done according to the protocol outlined in the Dako peroxidase-anti- peroxidase kit. N o r t h e r n blot: Total RNA was prepared using the g u a n i d i n i u m t h i o c y a n a t e - p h g n o l - c h l o r o f o r m procedure of Chomczynski and Sacchi (4). Poly(A)'mRNAS were subsequently extracted by the +use of oligo(dT) cellulose as described by Kingston (5). Poly(A) mRNAs were analysed by Northern blot as described elsewhere in detail (6). The CDNA probe for human UT is a 1 . 2 ~ b EcoRI fragment from human liver cDNA and was labelled with (J~P)dCTP by random priming (Random primed labelling kit; Boehringer Mannheim Biochemicals, U.S.A.). Results Mouse serum and extracts of mouse liver and kidney contain multiple substances of various molecular weight that inhibit trypsin activity (Fig.iA). Serum as well as liver and kidney extracts eluated of the anti-UT-IgG affinity column show inhibitory bands corresponding in size to that of purified UT, as depicted in Fig.lB.. H y d r o c o r t i s o n e and DOCA have different effects on the tissue and the serum levels of UT-like immunoreactive substance in the a d r e n a l e c t o m i z e d mouse. Hydrocortisone increased the liver and the serum levels of UT-like immunoreactive substance, while the renal levels did not change significantly. DOCA had no effect on tissue or serum levels of UT-like substance (Fig. 2). In the liver tissue, UT-like immunoreactivity was observed diffusely in the parenchyma, in contrast, in the kidney immunoreactivity was localized to the distal tubules, but not observed in the glomeruli, proximal tubules or the papillary ducts. A striking increase in immunoreactivity was observed in the liver of hydrocortisonetreated mice when compared to saline-injected mice, while no differences in renal staining could be detected in these two groups (data not shown). Northern blot analysis provides proof of specific h y b r i d i z a t i o n of the human UTcDNA probe to a single 1.3 kb transcript in the mouse liver which corresponds exactly to the mRNA size in human tissue (Fig.3). No signal could be detected with this cDNA probe in murine kidney, heart and lung.
1402
Urinastatin-like Substance in Mouse Liver
Vol. 50, No. 19, 1992
Discussion In humans, UT or UT-like substance is believed to be derived from human serum inter- ~ - t r y p s i n inhibitor (ITI), which is synthesized in liver tissue (7-9). S c h r e i t m 6 1 1 e r et al. (8,10) have suggested that ITI is a tight complex of three subunits. The smallest subunit in this large molecule is UT. In contrast, Salier et al. (9,11) contend that ITI consists of covalently linked light and heavy chains, and that UT is part of the light chain. In the present study, we have found that mouse liver possesses a protein with trypsin inhibitory properties that coincides in m o l e c u l a r w e i g h t with that of human UT. Furthermore, a 1.3 kb mRNA species was identified in mouse liver with a cDNA probe specific to human UT. This matches the size of the human UT
A 1
2
B 3
4
1
FIG.
2
3
1
Trypsin inhibitory activity demonstrated by using gelatin containing SDS-PAGE. A: lane i, UT (40 ng); lane 2, mouse liver extract equivalent to 40 mg of original wet weight; lane 3, mouse serum (0.5 ~i); lane 4, mouse kidney extract equivalent to 40 mg of original wet weight. B: lane I, UT (40 ng); lane 2, eluate of mouse liver extract from the anti-UT-IgG affinity column; lane 3, eluate of mouse serum from the a n t i - U T - I g G affinity column; lane 4, eluate of mouse kidney extract from the anti-UT-IgG affinity column.
4
vol. 50, No. 19, 1 9 9 2
Urinastatin-like Substance in Mouse Liver
1403
transcript which encodes a 40 kDa polypeptide chain found in human liver (9). This result supports the concept that ITI with its molecular w e i g h t of 150-160 kDa is in fact not a single chain protein but rather that UT is an integral component of ITI. In addition, the UT-like immunoreactive substance with trypsin inhibitory activity in the mouse liver, kidney and serum was found to have a nearly same molecular weight as that of UT. UT and UT-like substances are thought to be released by d i s s o c i a t i o n of the subunits of ITI w h i c h may possibly involve limited proteolysis of ITI. In vitro studies have shown that UT is released from ITI by proteolytic enzymes, such as granulocyte elastase (12), trypsin (12,13), chymotrypsin ( 1 3 ) and kallikrein (14) as well as by acidic conditions (i0).
12
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2
Changes in the levels of urinastatin-like substance in the adrenalectomized mice. Twenty-four hours after the a d m i n i s t r a t i o n of the drugs or vehicle, serum and tissue extracts of liver and kidney were prepared. UTlike immunoreactive substance was determined by the sandwich enzyme immunoassay. Numbers of d e t e r m i n a t i o n are shown in parenthesis. Statistical analysis were performed by Student's t-test. *P<0.05 HC: h y d r o c o r t i s o n e DOCA: d e o x y c o r t i c o s t e r o n e acetate
1404
Urinastatin-like Substance in Mouse Liver
Vol. 50, No. 19, 1992
In humans, the a d m i n i s t r a t i o n of g l u c o c o r t i c o i d s enhances the e x c r e t i o n of UT (15). This finding was r e p l i c a t e d in mice (i): adrenalectomy decreased urinary excretion of the UT-like immunoreactive substance, and administration of ACTH or hydrocortisone increased the excretion of this substance. Furthermore, the mineralocorticoid DOCA did not produce the same effect (i). In the present study, hydrocortisone increased urinary e x c r e t i o n of UT-like substance as well as liver and the serum levels of this protein, whereas DOCA did not. Mouse kidney tissue also contains a UT-like i m m u n o r e a c t i v e substance (16), and its level in h y d r o c o r t i s o n e - u n t r e a t e d mice is about o n e - f i f t h of that in the liver tissue. The level of i m m u n o r e a c t i v i t y in the kidney tissue did not appear to be affected by the a d m i n i s t r a t i o n of hydrocortisone. Using equal amounts of p o l y ( A ) - R N A s from several tissues, a single m R N A band of the a p p r o p r i a t e size could
kb 7.4-5.3-FIG.
2.8-1.91.64 - 1.3 kb 1.00.60.40.3-
3
N o r t h e r n blot analysis of mouse liver RNA. Poly(A) + RNAs in the liver tissue was prepared, and 1.2 ~ g of mRNA sample was loaded on a 1 % agarose gel containing 6 % formaldehyde, and run at 1 V/cm for 16 hr. Following capillary transfer of mRNAs to a Gene Screen Plus nylon membrane, the filter was probed with a random-primed human UT probe and exposed to film for 2 days. Size of the UTmRNA was calculated with RNA molecular weight markers shown in left side.
Vol. 50, No. 19, 1 9 9 2
Urinastatin-like Substance in Mouse Liver
1405
be clearly d e m o n s t r a t e d for the liver with the human UTcDNA probe, while no such band could be detected for the renal tissue. This observation is reminiscent of a finding by Salier and his associates in baboon tissues and human cell lines that the mRNA for the light chain of ITI (which these investigators believe to be a UT precursor) is found only in liver and not in other tissues, such as the kidney (17). The fact that pronounced increases in UT-like immunoreactivity could be detected in the liver and serum, while renal staining was virtually unchanged despite enhanced urinary excreation of this protein lends further support to the concept that the liver is the primary and probably sole site of UT synthesis. It is not clear whether ITI is actively dissembled in the distal tubule cells or whether dissociated UT is somehow taken up and possibly modified before excretion into the urine. Overall, the biological function of UT remains quite puzzling: a tumor-associated trypsin inhibitor which is identical to UT acts as a growth factor for endothelial cells in culture (18); the precursor of amyloid B-protein in Alzheimer's disease, has an amino acid sequence which is nearly 50 % similar to the inhibitory domain of UT (19), and trypsin bound to UT readily dissociates in the presence of e~-proteinase inhibitor or e2-macroglobulin to form a more ~table complex with this antiproteinases than does UT (20). We previously reported that amongst urine from dogs, cats, rabbits, guinea-pigs, rats and mice, only mouse urine contains a UT-like immunoreactive substance with trypsin inhibitory activity (i). The present experiments indicate that mouse liver contains a gene transcript that shows specific hybridization with a human UT probe and has a comparable size on N o r t h e r n blot. Whether the UT-like substance in mouse tissue is identical or highly homologus to the human UT requires further study, however, the present finding is convincing evidence that mouse is a suitable species for research on the biological functions of UT or UT-like substances. Acknowledgements The cDNA probe for human UT was generously provided by D r . W . G e b h a r d and Prof. K.Hochstrasser of the Ludwig-Maximilians University, M~nich, Germany. References i. T. SHIKIMI, S. SUZUKI, M. TAKAHASHI and H. KANETO, Scand. J. Clin. Lab. Invest. 50 1-8 (1990). 2. J.S. HANSPAL, G.R. B-~SHELL and P. GHOSH, Anal. Biochem. 132 288-293 (1983). 3. M. STEFANINI, C. DE MARTINO and L. ZAMBONI, Nature (London) 216 173-174 (1967). 4. P. CHOMCZYNSKI and N. SACCHI, Anal. Biochem. 162 156-159 (1987). 5. R.E. KINGSTON, Current Protocols in M o l e c u l a r Biology, F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl (eds) Vol.2, pp. 4.5.1-4.5.3., Greene P u b l i s h i n g Associates and Wiley Interscience, New York (1987). 6. D.M. STONE, T. WESSEL, T.H. JOH and H. BAKER, Mol. Brain Res.
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Urinastatin-like Substance in Mouse Liver
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8 291-300 (1990). 7. J.F. KAUMEYER, J.O. POLAZZI and M.P. KOTICK, Nucl. Acids. Res. 14 7839-7850 (1986). 8. T. SC~-REITMULLER, K. HOCHSTRASSER, P.W.M. REISINGER, E . W A C H T E R and W. GEBHARD, Biol. Chem. H o p p e - S e y l e r 368 963-970 (1987). 9. J. BOURGUIGNON, M. DIARRA-MEHRPOUR, R. SESBOUE, M. FRAIN, J.M. SALA-TREPAT, J.P. MARTIN and J.P. SALIER, Biochem. Biophys. Res. Comm. 131 1146-1153 (1985). 10. W. GEBHARD, G. LEYSATH and T. SCHREITMULLER, Biol. Chem. H o p p e - S e y e r 369 Suppl. 19-22 (1988). ii. J-P. SALIER, M. DIARRA-MEHRPOUR, R. SESBOUE, J. BOURGUIGNON and J-P. MARTIN, Biol. Chem. Hoppe-Seyer 369 Suppl. 15-18 (1988). 12. T. DIETL, W. DOBRINSKI and K. HOCHSTRASSER, Hoppe-Seyler's Z. Physiol.Chem. 360 1313-1318 (1979). 13. P. REISINGER, K. HOCHSTRASSER, G.J. ALBRECHT, K. LEMPART and J-P. SALIER, Biol. Chem. Hoppe-Seyler 366 479-483 (1985). 14. G. BRETZEL and K. HOCHSTRASSER, Hoppe-Seyler's Z. Physiol. Chem. 357 487-489 (1976). 15. H.J. FAARVANG, Acta Pharmacol. Toxicol. 19 293-304 (1962). 16. T. SHIKIMI and S. SUZUKI, Biol. Chem. Hoppe-Seyer 371 991-997 (1990). 17. J.P. SALIER, M. DIARRA-MEHRPOUR, R. SESBOUE, J. BOURGUIGNON, R. BENAROUS, I. OHKUBO, S. KURAUCHI, K. KURAUCHI and J.P. MARTIN, Proc. Natl. Acad. Sci. USA 84 8272-8276 (1987). 18. W.L. MCKEEHAN, Y. SAKAGAMI, H. HOSHI and K.A. MCKEEHAN, J. Biol. Chem. 261 5378-5383 (1986). 19. R.W. CARRELL, Nature (London) 331 478-479 (1988). 20. B-H. JONSSON and K. OHLSSON, H o p p e - S e y l e r ' s Z. Physiol. Chem. 365 1403-1408 (1984).