synthesis of hemopexin and cysteine protease inhibitor is coordinately regulated by hsf-ii and...

Vol. 146, No. 3, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 14, 1987 Pages 1218-1226

SYNTHESISOF HEMOPMIN~CYSTEINEPRUIEASE INHIBITOR IS CKIRJXNATELYRECUEATEDBYHSF-IIAIW INI'ERFFRDI+82 INRWHEPAICMACELLS

Heinz Baumann* and Ursula Muller-%erhard§ * Department of Molecular and Cellular Biology,

Roswell Park Memorial Institute , Buffalo, New York 14263

§ Departments of Pediatricsr Pharmacology and Biochemistry, New York Hospital-Cornell University Medical College,

525 East 68th Street1 New York1 New York 10021

Received June 19, 1987

SUMMARY: Rat hepatoma (H-35) cells respond to hepatccyte-stimulating factors by increased expression of major acute phase plasma proteins. The synthesis of henopexin is stimulated lo-fold by either hepatocytestimulating factor-II of human squamous carcinoma cells or hepatocyte-stimulating factor/interferon+2 of activated human blood monocytes. The hormone specificityr time course and dose-dependence of hemopexin regulation is closely correlated with that of cysteine protease inhibitor. The coordinate expression of hemopexin and other type II acute phase proteins suggests the existence of corrmon molecular regulatory mechanisms. 0 1987 kademic Press, 1°C.

In the ratr acute systemic injuries cause a 2- to loo-fold increase in the

levels of acute phase plasma proteinsr including 2 -acid glycoprotein, 1

haptoglobin, complement C3 (C3)r fibrinogenr hemopexin (Hx)r cysteine protease

inhibitor (CPI)r contrapsinr ~1 -antitrypsi.n and c1 -macroglobulin (1,2). These 1 2

proteins are produced by the liverr peak levels are reached within 24 to 36 hr

and their synthesis is controlled by dexamethasone (Dex) and by hepatocyte-

stimulating factors (HSFs) (3r4). A major source of HSFs are keratinocytes (5)

and activated monocytes (4r6). Human squamous carcinoma (COW16) cells produce

2 structurally distinct HSF form.% HSF-I and HSF-II (7). HSF-II a protein with

Mr = 18r500 and pI 5.5r induces predominantly ~1 -acid glycoprotein, hafl-l&in 1

and c3. HSF-IIr a glycoprotein with Mr = 34rOOO and neutral to basic charge,

ABBREVIATIONS: COLQ-161 human squamous carcinoma1 cell line; COL&16 CMI conditioned medium of human squamous carcinoma1 cell line; CPIr cysteine protease inhibitor; C3r corrplement C3; Dexr dexamethasone; HSFI hepatocytestimulating factor; Hxr hemopexin; IL-lBr recombinant human interleukin 16; INF-B2 (= BSF-11)1 reconbinant interferon B2; TNFl tumor necrosis factor.

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. AN rights of reproduction in any form reserved. 1218


mainly induces acute phase protease inhibitors. Both HSF forms are

structurallyr imologically and functionally distinct from human interleukin 1

(IL-11 and tumor necrosis factor (TNEY.

Activated hunan peripheral blood monocytes releaser besides IL-U? and TNFI

one HSF form with Mr = 29rOOO and p1 5.1 (8). This factor's activityt but not

its structurer reseribles that of HSF-II from COLD-16 cells, and it is identical

with reCOnbiIX& interferon-B chain (IFN-62) (9). 2

A Reuber H-35 subliner like the phenotypically unstable (10) cultures of

primary cultures of adult rat hepatocytes (5,6), is responsive to regulation of

all major acute phase proteins (7). Among all known hepatoma cell lines of any

speciesr the H-35 cells are unique in as much as the extent of the induction of

Hx is equivalent to that observed in Y&Q in rats (3,ll-14). This property

allowed us to unambiguously describe the factor-specificity of Hx expression and

to document the similarity of its regulatory pattern to that of CPI.

and &QQ& .&I,& The H-35 cells (7,151 were maintained in Dulbecco's minimum essential medium containing 10% fetal calf serum.

1 m Crude preparations of HSFs were the conditioned media of COD16 cells (COLO-16 CM). The s CM (7) I was 2 1 x 10

pific activity of HSF-111 isolated from serum-free COLD-16 HSF units/ml. One HSF unit is defined as the concentra-

tion of HSF inducing al-antichynotrypsin in Hep G2 cells to one third maximal level (6).

Conditioned medium of I&S-activated human peripheral blood rronxytes was the source of monocytic HSF (= IF&!321 (4,5,9). HSF separation from Z-18, was achieved by subjecting 1 ml of this medium to HPIC on TSK-3000 in Tris-buffered saline and collecting 0.4 ml fracti ns.

Recombinant human X-16 (2 x 10 8 units/ml) was from Immunex Corporation and recombinant IFN-62 (DSF-II) (2 x 105 units/ml) a gift of Drs. Hirano and KishiKoto (16).

_ . and A&&z&a of P&XI@ ProI- H-35 cell monolayers (80% confluent) were treated for 40 h with the test

factors in medium containing 2% fetal calf serum (7). Hedia were dialysed for 6 h against 25 til !@14K0 and lyophilized.

2 Plasma protein levels in resuspended

madium aliquots were m asured by rocket @nnunoelectrophoresis and the data expressed in ug secreted per 48 h and 10 cells (7r17). The induction of proteins in response to the factors was also assessed in primary cultures of rat hepatccytes prepared from the liver of a 4 month-old female AC1 rats (18). The cells were plated into collagen-coated 6-well plates in DMEM containing 10% fetal calf serum. Treatments were started after a recovery period of 18 h. Test media (1 ml) were replaced after 24 h. Plasma proteins secreted during6the second 24 h culture period were expressed in ug secreted per 24 h and 1 x 10 cells.

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A crude preparation of HSFsr i.e. conditioned medium of COD16 cellsf

induces Hx in H-35 cells maximlly between 8- to lo-fold (Table I). This effect

is independent of glucocorticoidsr e.g. Dex. Moreoverr Dex by itself causes no

significant rise in the basal level of Hx. Concomitant with increased Hx

synthesisr the synthesis of CPI (representative of type II acute phase proteins)

and C3 (representative of type I acute phase proteins) are increased by a factor

of 20. The regulation of CPI differs from that of I& and C3 by the synergistic

actions of Dex and COLD-16 factors. NSF-11 purified from COLO-16 CX induces Hx

to the same level as non-fractionated medium (Table I). This finding suggests

that no additional COLCk-16 cell-derived factors are required for maximal

response.

TABLE I

Effect of HSFs on plasma protein production by H-35 cells

Treatment Secretion48 h B 106 a

Hx CPI c3

None 0.5 0.01 0.01

DeX 0.7 0.04 0.01

COIQ-16 CM 4.1 0.21 0.18

COLO-16 CM + Dex 3.8 0.43 0.13

HSF-II 4.6 0.15 0.09

SF-11 + Dex 3.7 0.44 0.09

DeX 0.8 0.01 0.03

IN+82 @SF-II) + Dex 8.6 0.55 0.06

In two separate experiments, duplicate monolayers of H-35 cells were treated for 48 h with DMEM containing 2% fetal calf serum aloner or with 1 uM Dexr l/10 diluted COLO-16 CMr purified HSF-II of COLD-16 cells (50 units/ml), or recombinant IW-62 @SF-III 50 units/ml). The amounts of Hxr CPI and C3 were determined by rocket imnunoelectrophoresis.

1220


, 0 l A

0 Hemopexin 40- 0 CPI

300- 3- A C-3

I I

h

I 24 36 46

2

Hours of Treatment

wl. Time course of the induction of acute phase proteins in H-35 cells. Duplicate monolayers of H-35 cells in 6-well cluster plates were treated with HSF-II in DMESI containing 2% fetal calf serum and 1 uM Dex. Addition of the test factors were coordinated such that all cultures simultaneously reached the indicated lengths of treatment times. Four h prior to reaching the end pointr the medium was removed. The cells were washed 3 times and incubated for 4 h in fresh medium. The amounts of secreted proteins during the last 4 h period were determined by rocket iranunoelectrophoresis.

The fact that HSF-II fully accounts for maximal induction of CPI but not of

C3r supports our recent proposal (7) that FJx is a type II acute phase protein.

Additional supporting evidence is provided by results of kinetic studies. The

rate of synthesis of several type II acute phase proteins in H-35 cells

gradually increases, after an initial lag of several hours, and reaches a

maxin~~~ level after 48 h of HSF treatment (7). Type I acute phase proteins, on

the other handr reach their maximal synthesis rates after 24 h. Measurements of

Hx synthesis rates during HSF-II treatment revealed that the time course for its

induction is essentially the same as that for CPI but differs significantly from

that for C3 (Fig. 1).

The synthesis patterns for Hx and CPI correlate not only temporally but also

regarding dose-dependency of hormone addition. Cultures incubated with serially

diluted HSF-II demonstrate that Hx and CPI synthesis are equally affected (Fig.

2). Maximal cell response is accomplished with 100 units/ml. At that

concentrationr the magnitude of the induction of Hx ranges between lo-12 fold.

At concentrations of HSF-II above 500 units/ml, an inhibitory effect occurs~

although no change in cell viability is detected.

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0 1 10 100 1,000 10,000

HSF Units

r Dose-dependence of the induction.of acute phase proteins in H-35 . Duplicate monolayers of H-35 cells m 6-well cluster plates were treated

for 48 h with medium containing the indicated units HSF-II per mlf 2% fetal calf serum and 1 uM Dex. The amounts of EIXZ CPI and C3 secreted during the 48 h stimulation period were determined by rocket immnoelectrophoresis.

!l?hese results strongly suggest that in H-35 cells the expression of Hx and

CPI are coordinately regulated by HSF-II.

Considering that H-35 cells represent transformed liver cells and might

display an abnormal HSF respOnser the effect of HSF was also determined in

primary cultures of adult rat hepatocytes (Table II). Although rat hepatocytes

differ from H-35 cells in their higher basal and stimlated rates of plasm

protein productionr the qualitative response patterns are virtually identical.

Induction of Hx and CPI by unfractionated COL&mdiuxn is equivalent to that by

purified SF-II. Dex has no further enhancing effect on Bxr but as in H-35

cells, on CPI induction.

IL-1 induces in rat hepatocytes type II but not type 111 acute phase

proteins (7). Howeverr when IL-1 was applied at concentrations greater than 100

U/mlr a low but significant increase in Hx synthesis is detected. This finding

is exceptionalr since under the same conditions the synthesis of CPI (or of any

other type II protein) remains unaffected (Table II). The contribution of IL-1

to the regulation of Hx achieved by unfractionated CO-16 CM is, however,

insignificant due to the relatively low amount of IL1 activity produced by

these cells (5).

1222

. 146, No. 3, 1987 VOI.

A

0.4

0 @J

a? 0.2

0 ii -+----XT 20 30150 60 70

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Oh 6

Time after injection, min

1 T3 TX T5'

T5

LU

Tl'

vm 20 30150 60 70 80

Time after injection, min

Figure 2. Elution Profiles from RP-HPLC Purification of Tryptic Peptides of C7 d C7' Peptides C7 and C7' were cleaved with trypsin and 1 yophilized

!:yPtic peptides were resuspended in 30% acetic acid and applied to the Altex Ultrasphere C-18 reverse-phase column. Peptides were eluted with a linear gradient of O-60% acetonitrile in 90 minutes. Elution profile of the peptides from the trypsin cleavage of C7 (81) is shown in panel A and that of the peptides from the trypsin cleavage of C7' (83) is shown in panel B.

in the chromatogram of f33+Bl (Figure 1B) suggesting that C7' represents a 83

peptide that contains an amino acid substitution relative to Bl.

CNBr peptides C7 (Bl) and C7' (83) were cleaved with trypsin and the

tryptic peptides were isolated by reverse-phase HPLC (Figure 2). The elution

pattern from the trypsin cleavage of C7 contained 7 peaks representing five

different peptides, Tl-T5 (Figure 2A). The doublets Tl,Tl' and T5,T5' were

the result of incomplete cleavage of the Lys-Lys sequence at positions 338-339

of the Bi sequence (11). The amino acid composition of Tl (Table 1) corre-

sponded to the Ala-Lys at positions 337-338 of ~1 (11) and Tl' corresponded

to Ala-Lys-Lys at positions 337-339 of 81. The amino acid composition of T5'

(Table 1) corresponded to amino acids 340-354 of B1 (11) and T5 corresponded

to residues 339-354 of ~1 (11). The composition of peptide T3 corresponded

to amino acids 355-366 of ~1 (11); that of peptide T2 to amino acids

367-369; and that of peptide T4 to amino acids 370-374 (Table 1).

Peptides Tl, Tl', T3, T5, and T5' were present in the chromatogram of the

tryptic cleavage of peptide C7' (Figure 28). T2 and T4 were absent in the C7'

profile; they were replaced by a single peptide, TX (Figure 2; A vs. B). The

composition of TX (Table 1) isolated from C7' (83) was equal to the sum of

the compositions of T2 and T4 except that the Arg in T2 was replaced by CM-Cys

in TX.

The amino acid sequence of tryptic peptide TX, determined through 7 cycles

of the sequenator, corresponded to amino acids 367-373 of the B1 sequence (11)

except that CM-Cys had been substituted for the expected Arg at position 369

(Figure 3). The C-terminal Phe-374 was not recovered from the sequenator;

however, a single Phe was observed in the amino acid composition of TX

(Table I), indicating that the C-terminal Phe-374 was undoubtedly present in

the 83 subunit.

1230


0

=" i? 500

"0 T ; 400

AZ

3 \ 300

z

c; 200 .g

e

fii 100 rn

n 12 14 16 16 20 22 24 26 26 30 32 34 36

Fractions

w3. Inducing effect of HPLC-separated n-onccytic HSF activities. Four 1 ml aliquots of conditioned mediumof LPS-activated human blood monocytes were separated in parallel by HPLC. The corresponding HEJLC fractions of the 4 aliquots were Pooled and tested for their inducing effect after a lo-fold dilution with DMF,M containing 2% fetal calf serum and 1 uM Dex. The elution Positions of the molecular weights fK?r6ni) of standard proteins are indicated at the top.

preparation). iiecorrbinant IFN-B2 (assigned ESF-II (16))r when tested on primary

rat hepatocyte culturesl fully accounts for HSF activity with Nr = 321000 in

rronocytic conditioned medium. IFN-62 (BSF-II) addition to H-35 cells causes

maximal induction of all type II acute phase proteins. As shown in Table II

Experiment IIr the mgnitude of the induction of Hx and CPI by IF&B2 is equal

to that by COD16 and HSF-II.

We conclude from these results that Hx expression in rat liver cells is

coordinately regulated with other type II acute phase proteins by keratinocytic

HSF-II and monocytic HSF/IFW32.

One of the remarkable features of the hepatic acute phase response is the

coordinate increase of several plasma proteins (lr20~21). The regulatory

mechanisms mediating the coordination conceivably involve or depend on a) a

liver-specific hormsner b) a conmon intracellular secondary messenger systemr c)

separate signals activating a connnon mediator pathway! and/or d) conmon

cis-acting regulatory elements near or within the structural genes for the acute

1224


phase proteins. The possible role of each of these components are under study

in numerous laboratories. Progress in defining the regulatory mechanisms

depends on a suitable tissue culture system allowing verification of the

functional properties of the proteins under study. H-35 cells proved to be

extremely valuable in 2 major aspects. 1) The pronounced response of these

cells to purified factors revealed that the expression of subsets of acute phase

proteins, e.g. type I and type JIr depends on a single inducing ag'ent. And 2)

since a large number of plasma proteins is regulatd in these cells, the

coordinate expression and hormone specificity of the response can be defined.

The present study was limited to the measurement of protein levels in

culture media. In the next phase we will delineate at which level hemopexin

expression is regulated: at the level of transcriptionr rfRNA stability, and/or

translation? The process of secretion is probably not an important regulatorr

as secretion of Hx into the medium of prirary cultures of rat hepatccytes is as

fast as that of al'oumin and faster than that of transferrin (22). The

regulation of the synthesis of other rat type II acute phase proteins can occur

at different levels. CPI and fibrinogen apLpear to be strictly regulated at the

transcriptional level (191, while c1 2 -macroglobulin is predominantly regulated at

the post transcriptional levelr probably by m& stabilization (23).

Surprisingly, although separate regulatory mechanisms are involved, they appear

to be coordinated to such a degree as to increase mRNA concentrations and

protein production in the acute phase response.

Acknowledgments

We are greatly indebted to Dr. J. Gauldie @i&laster University) for providing fractionated conditioned medium of human peripheral blocd monocytes and Drs. T. Birano and T. Kishiroto for recombinant BSF-II. We thank G.P. Jahreis and J. Bordonaro for technical assistance and M. Held and S. Seeley for secretarial work.

This work was supported by NC1 grant CA26122 and NIH AM30203. H.B. is a recipient of an AHA Established Investigator Award.

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synthesis of hemopexin and cysteine protease inhibitor is coordinately regulated by hsf-ii and...

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