Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types

Vol.

172,

No.

October

30,

2, 1990

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1990

NEUTRAL ENDOPEPTIDASE OSTEOBLASTIC Fred

E. Indigl,

IS HIGHLY EXPRESSED ON

Benayahu', Adina Fried:, and Shmaryahu Blumbergi"

Shlomo

Wientroub2*3

of Molecular Medicinei and Department of Histology Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel

Department

September

(EC 3.4.24.11)

620-626

CELLS AND OTHER MARROW STROMAL CELL TYPES

Daphna

Sackler Institute and Cell Biology',

Received

AND

of Orthopaedics3, Tel Aviv Tel Aviv 64239, Israel 4,

Medical

Center,

1990

A series of cultured mouse marrow stromal cell lines of different characteristics were examined for their neutral endopeptidase using the highly selective chromophoric substrate of the (NEP) activity enzyme, 3-carboxypropanoyl-alanyl-alanyl-leucine 4-nitroanilide. All the cell lines tested contained appreciable amounts of NEP activity, but the cells expressing an osteoblastic phenotype, MBA-15, showed the highest levels. Other non-stromal osteoblastic cell lines, MC-3T3-El, ROS 17/2.8 and cells derived from mouse long bone explants, were also rich in NEP. A fourfold stimulation of NEP activity was observed when certain MBA-15 clones were cultured in the presence of la,25-dihydroxyvitamin D3. Since NEP is an effective cell-surface endopeptidase, it may play a role in the dynamics of bone formation, via interaction with biologically active polypeptides. c 1990 RcademlcPE55, 1°C. SUMMARY:

phenotype

Neutral enzyme

endopeptidase

present

in mammalian system the

including

terized

of

bacterial

cells

it

* To whom reprint 0006-291X/90

of

leukemia

proteinase

very

may

P, atria1

efficient play

requests

antigen [6].

of

(CALLA,

endopeptidase

a role

in marrow

should

be addressed.

620

cell

ecto-

the

immune

with [4,

that

of

the

is impliincluding

and interleukin-16

present

development

basic

well-charac-

The enzyme peptides

factor

of

51 and is

NEP shares

[7-g].

and is

an

distributed

in

CD 10)

with

a variety

natriuretic

is

identical

Catalytically,

properties

$1.50

and

tissue

thermolysin

and regulation

(NEP)

and is widely

human NEP is

species

Substance

As NEP is a [3],

neutral

types brain

and mechanistic

metabolism

enkephalins,

kidney,

sequence

in different

specificity

in the

[lo].

acid

enkephalinase

or

of many cell the

lymphoblastic

conserved

cated

surface

The amino

common acute

elements

the

tissue

[l-3].

highly

EC 3.4.24.11

on the

on bone marrow and function

by

Vol.

172,

No.

2,

modulating sence should

since

stromal

stromal

Although

nitude

all

higher

observed

on other that

the

lines

cells

enzyme

are

develop

NEP activity

the

other

with

osteoblastic

into

the

of

the

of

cultured

characteristics

[ll,

amounts

had 1-2

Such high dynamics

precells,

committed

series

appreciable

characteristics, to

the

functional

of a

phenotype cells.

may be important

respect

constituents

phenotype

contained

than

this

important

an osteoblastic

MATERIALS

In

COMMUNICATIONS

on lymphohematopoietic

of different

tested

expressing NEP levels

only

cells

the

lines

RESEARCH

substrates.

and may also

cell

cell

cells

BIOPHYSICAL

not

cells,

microenvironment

marrow

AND

of polypeptide on stromal

on their own. In this study we determined

activity,

bility

activity enzyme

marrow

mouse 121.

the

be considered

bone cells

the

of

BIOCHEMICAL

1990

of

orders

levels raise of bone

of

NEP mag-

of NEP, also the

possi-

remodeling.

AND METHODS

Materials. 4-Nitrophenyl phosphate disodium hexahydrate and 2-amino-2methyl-1-propanol were from Sigma. Phosphoramidon was from Peptide Institute (Japan); 3-carboxypropanoyl-alanyl-alanyl-leucine 4-nitroanilide (SucInc. Ala-Ala-Leu-NH-Np) and Streptomyces griseus aminopeptidase (SGAPI) were obtained as previously described (13, 14). Cell Culture. Mouse bone marrow stroma-derived clonal cell lines, MBA1.1.1 (fibroblast), MBA-2.1 (endothelial), MBA-13.2 (fibroendothelial), MBA15 (osteoblastic) (11, 12) and the MBA-15 clones (MBA-15.4, MBA-15.6, MBA15.30, MBA-15.31 and MBA-15.33) (15) were used. Other osteoblast cell lines used were: ROS 17/2.8, originating from rat osteosarcoma (16); MC-3T3-El, nontransformed cloned osteoblast from mouse calvaria cell line (17) and (collagenase released) osteoblast cells obtained from mouse long bone (after 5 passages) (18). Stock cultures were maintained in Dulbecco's Modified containing 4.5 g glucose/liter and supplemented with 10% Eagle's Medium, fetal calf serum (Bio-Lab Ltd., Jerusalem). Cultures were passaged once weekly by removal with a teflon policeman and dispersion in fresh medium. ROS 1712.8 and MC-3T3-El cells were passaged once weekly by trypsinization. All cultures were incubated at 37°C in a humidified atmosphere of 10% COz in air and medium was changed every 3 days. Cells were seeded at 5x104 cells/ in 30 mm plates. The cells were harvested at confluence (5-8 days), plate rinsed twice with phosphate buffered saline, scrapped off and transferred to 0.25 M sucrose. The cell suspension was sonicated on ice for 30 s at full power, stored at -20°C and used within a week for enzyme activity and protein determinations. Preparation of cell membranes and intact cells. MBA-15 cells from five 90 mm tissue culture plates at confluence and sub-confluence were collected, suspended in 20 ml 0.25 M cold sucrose and dispersed by pipetting several times through a 5 ml pipette. Fifteen ml of the suspension was sonicated until no intact cells remained (6x30 s pulses, full power). Sonicate was centrifuged at 450 x g for 10 min and the supernatant centrifuged at (81) 100,000 x g for 80 min. The pellet (Pz) was resuspended in 37.5 mM Tris-HCl/ 112 mM NaC1/62.5 mM sucrose, pH 7.5, Triton X-100 was added to a final concentration of 1% (v/v) and the mixture was stirred for 15 min at room temperature. The mixture was centrifuged at 100,000 x g for 60 min yielding a solubilized membrane fraction (S3). The fractions were assayed for their NEP activities. The remainder of the cell suspension (6 ml) was pelleted by low speed centrifugation (450 x g), resuspended as above, counted and assayed. 621

Vol.

172, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Enzymic activities. NEP activity of cell sonicates was assayed by the two-stage assay using the synthetic substrate Sue-Ala-Ala-Leu-NH-Np supplemented with SGAPI essentially as described (13). Assays were performed with 0.4 mM substrate in 112 mM NaC1/37.5 mM Tris-HCl/l mM CaClz/62.5 mM sucrose, pH 7.5, 23°C in the presence of 26.7 pg/ml SGAPI (14) in a volume of 0.2 ml. NEP activity of membrane fractions and intact cells was assayed at 29°C. Selectivity of the NEP reaction was assessed by inhibition with 1.8 @i phosphoramidon. Alkaline phosphatase activity was assayed with 2 mM 4-nitrophenyl phosphate at 37"C, in 0.1 M 2-amino-2-methyl-1-propanol and 2 mM MgClz essentially as described (19). Protein determinations. Protein was determined by the method of Bradford using bovine serum albumin as standard or by the method of Lowry (Zl), human serum albumin as standard. Enzyme assays and protein determinations were performed with microplates (Nunc Immuno Plate Maxisorp F96 or Flow Labs.) using a Kinetic Microplate Reader (Molecular Devices Corp.). (20) using

RESULTS NEP activity

of

Screening teristics

stroma-derived of

lines

stroma-derived

tested

displayed

nmoles/min/mg

protein

1.8

fl

MBA-15

cell-lines fibroblast an of

activities.

Table

exhibiting

fibroblast

specific orders

of

Cell

line

MBA-1.1.1 MBA-2.1 MBA-13.1 MBA-15

of

a Activity

% Inhibited by 1.8 pM Phosphoramidonb 97 82 53 97

measured

with

cell by

phosphoramidon

cultured

cell

levels

of

or both

phosphatase,

a different

cells,

pattern

MBA-1.1.1

also

mouse marrow

Alkaline phosphatase activityC (nmoles/min/ mg protein)

NEP

Activity (nmoles/min/ mg protein)

17.5fO.6 3.9 79.2t2.7 7.820.2

Sue-Ala-Ala-Leu-NH-Np

with Streptomyces aminopeptidase. Values (mean + S.E.) experiments, each representing the average of triplicate b Based on 4 experiments each carried out in duplicate or c Based on 2-4 experiments carried out in triplicate.

622

0.4-29.1

NEP

Alkaline

6.3 2.5 0.4 29.1

the substrate

of

(MBA-2.1)

showed MBA-15

the

osteoblastic

the

(MBA-13.5).

24.11 activity stromal cell lines

3.OkO.2 0.820.07 3O.Of2.2

the

endothelial

osteoblastic

charac-

all

inhibitable NEP,

of than

phenotype,

6.5t0.9

range

of

Net

Activitya (nmoles/min/ mg protein)

that

the

NEP level

magnitude

properties

to the

endopeptidase

Neutral

in

(MBA-l.l.l),

cell

established

revealed

inhibitor the

of osteoblastic

addition

of different

Sue-Ala-Ala-Leu-NH-Np,

Remarkably, by 1-2

marker

lines

NEP activity, NEP activity

the

l-4).

was higher

In

I.

their hydrolyzed

and endothelial

established

cell

for

of of

columns

I,

lines

appreciable

concentrations

(Table line

22)

12,

(11,

cell

are

supplemented based on 4

determination. triplicate.

Vol.

BIOCHEMICAL

172, No. 2, 1990

showed

a high

lowest

NEP level

line

alkaline among

phosphatase Five

terms

of

the

Location

alkaline

two enzymes

did

centrifugation

membrane total

stromal

that

the

cell

to

cell

NEP activity

lymphocytes Effect

this

1,25(OH)zD3

3 days

gical

concentration

Under

the

High

expression

its

clones

phenotypical several

3.2

effects

expression

raise property

non-stromal

the

were

the

membrane-

in

possibility

fraction

debris

was by

at

100,000

of (S3)

of

low

present

fraction

were

this

in

of

the

95%

of

following solubilized

of NEP present performed

dishes. to

on

These

nmoles/min

on the

an

assays

per

the

acute

of

lo6

MBA-15 revealed

cells.

OR

NEP activity

enriched

a

present

lymphoblastic

agents

exposed

to

leukemia

in NEP.

II the

that

growth

clones

of

MBA-15

the

added

D3

24

and the

cells

1,25(OH)~D3

at

NEP levels

phosphatase

in cell

was serum

shows

active

la,25-dihydroxyvitamin

derivative

D3

h

after

were

har-

physiolo-

of the

clones

four-fold.

were

hardly

changed.

levels

cells

of NEP on the that

of osteoblasts. lines

clones

6%. Treatment

of NEP activity,

Table alkaline

cell

five

centrifuged

character

charcoal-stripped

levels

(2.7-118.4

the

cell

only

to be highly

of NEP on osteoblastic high

disparate

levels

the

(10 nM) enhanced

The unusually

phosphoramidon.

fl

NEP activity

soluble

culture

known

The vitamin

same conditions

1.8

in solubilization

and on common

post-treatment.

9.6-

were

is comparable

types

containing

of

of

total

assays

the

MBA-15.33,

treatment.

in medium

range

protein.

cell

evaluating

and

in the

NEP activity

ectoenzyme

activity

on the

(1,25(OH)zD3) plating

in the

to approx.

[13]

also

demonstrate

contained

on NEP expression

towards

MBA-15.30

alka-

were

a membrane

(Si)

resulted

remained

from

two cell

and differentiation cells,

X-100

intact

cells

[231,

of

(Sz)

the

to

the

x g. The specific

amounts

basis

As a step

vested

1% Triton

lines,

on human neutrophil

of

(15)

for

removal

94% of

recovered

cell

After membranes

demonstrate

suspension

level

the

line

more

NEP activity,

was 12 nmoles/min/mg

further

having

membranes

and in order

the

which

cell

activities

contained

100,000

preparation

To cell

at

cell

supernatant

NEP activity

membrane marrow

the

were

of

measured

(PI),

with

centrifugation

highest

by 397% with

sonicates.

(Pz)

fraction

cells,

parallel.

enzyme

cell

whereas

the

of NEP activity,

order

not

of the

The precipitate mixture

MBA-13.5

MBA-15

phosphatase the

on the

surface

MBA-15

the

inhibitable and

from

speed x g.

the

for

character

prepared

showed

from

amounts

protein)

a cell

associated

the

high

of NEP activity

NEP is

derived

protein,

obtained

nmoles/min/mg

tested,

the

RESEARCH COMMUNICATIONS

1, column 5).

showed

nmoles/min/mg

but

level,

cells

clones

and all

The values

the

(Table

different

examined 80.9

phosphatase

AND BIOPHYSICAL

high Table

with 623

those

osteoblastic

MBA-15

NEP levels III of

may serve

compares the

MBA-15

the

NEP cells.

cells

and on

as a novel levels All

of osteo-

Vol.

172,

No.

2, 1990

Table

II.

BIOCHEMICAL

Effect

of

AND

1,25(OH)zD3 cultured mouse

in

MBA-15.30

MBA-15.33

RESEARCH

on neutral endopeptidase bone marrow cell linesa

1,25(OH)zDx MBA-15-derived clone

BIOPHYSICAL

(nM)

NEP activityb (nmoles/min/ mg protein)

0 0.1 10 0 0.1 10

6.2kO.4 6.7kO.4 31.4fO.7 4.7fO.3 7.2i1.3 19.1t2.6

COMMUNICATIONS

24.11

Alkaline activity minlmg

expression

phosphatase (moles/ protein)

5.5to.4 4.9kO.4 4.6tO.2 4.8iO.4 4.0+0.4 5.9kO.5

a Values (mean + S.E.) are based on 3-4 experiments each representing average of triplicate determination. b All activities are inhibited by 296% with 1.8 @l phosphoramidon.

blastic

cells

cells

showing

highest

tested the

level

showed lowest

(160

levels

generally

high

but

fered

from

observed

of

NEP

nmoles/min/mg

nmoles/min/mg

also

that

high

(5.0

protein).

the

order

for

NEP (Table

activity,

protein)

the

with

ROS 1712.8

and MC-3T3-El

Alkaline

phosphatase

of activities

in the

cells

the

levels

different

were

cells

dif-

III>.

DISCUSSION The

present

study

amounts

in mouse

various

stromal

cells

having

extends line to

to derived

1,25(OH)zD3

in processes

Table

Cell

III.

cell

several from

other mouse

long

exposure triggered

Neutral

NEP cell

is

established

osteoblastic III).

(Table by the

(Table

vitamin

endopeptidase osteoblastic

D3

24.11

protein)

putative

especially latter

enriched

cell

lines,

and a cell

in NEP of

activity the

enzyme

derivative.

activity phenotypea

on cultured

cell

lines

with

Alkaline phosphatase activity (nmoles/min/mg protein) 7.8kO.2 N.D.d 104.6t15.5 108.923.5

S.E.)

are based on 3-4 experiments determination. with 1.8 @l phosphoramidon. 1.8 @l phophoramidon.

624

each

in

characteristic

participation

30.0+2.2b 160.0+6.gb 49.1t1.8 5.0k0.2c

bone

in appreciable

representing

The response

may indicate

II)

present

lines

bone

NEP activity (nmoles/min/mg

a Values (mean + average of triplicate b Inhibited by 297% c 86% inhibited with d Not determined.

that

stroma-derived

and that the enzyme is types phenotype (Table I). This

osteoblastic

line

MBA-15 MC-3T3-El Mouse long ROS 17/2.8

demonstrates

bone marrow

representing

the

Vol.

172,

No.

2,

While

with

attributable

caution,

a

variety

molecular

of

results

the

present

is

present

surface

surface

modulation

of

peptidase

also

are

mass peptides [3]

here

action

on the

also

with

its been

be roles

of

hydro-

on a higher

reported

(10).

expression

immune

stroma-derived

the

action

may reflect

in the

should

capable

remarkable

demonstrated,

polypeptide found

and its

lines

consistent but

has

COMMUNICATIONS

cell

endopeptidase

interleukin-lB,

cells

RESEARCH

on

findings

in bone marrow

stroma-derived the

a cell

BIOPHYSICAL

obtained

low molecular

mass polypeptide,

enzyme

AND

of

to NEP. NEP is

lyzing

in

BIOCHEMICAL

interpretation

regarded

ferent

1990

The

on dif-

an important system.

cells

is

role

Another

cell-

aminopeptidase

N

(unpublished). Certain

tissues

cells,

are

and

efficient

especially

specialized

and thereby

bolizing

peptides

factor

and other

philes,

NEP is

as fMet-Leu-Phe peptides

cell

enriched

peptidase

osteoblastic

and

thought (10).

cells involved

as the

of

infiltrated

may in the

is

to act

possible

that

be important processes

the

in the of

NEP is

substance the

bone

and neutrophile

to

peptidase

through

to be required It

kidney,

the

enkephalins,

kidney

in order

In the

key component

peptides

as the

in NEP, probably functions.

the such

such

types,

the

rapid

major

endo-

machinery, P, atria1

kidney

upon

chemotactic levels

meta-

natriuretic

(24).

high

turnover

perform

On

neutro-

peptides

such

of NEP present of

peptides

on

and poly-

remodeling.

ACKNOWLEDGMENTS We are grateful to Prof. Dov Zipori, Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel for the MBA cell lines. This work was supported by grants from the Israel Academy of Sciences and Humanities - Basic Research Foundation (S.W.) and the Moise and Frida Eskenasy Institute for Cancer Research (S.B.).

REFERENCES 1.

2. 3. 4. 5. 6.

7. 8. 9.

Malfroy, B., Swerts, J.P., Guyon, A., Roques, B.P. and Schwartz, J.C. (1978) Nature 276, 423-526. Gee, N.S., Bowes, M.A., Buck, P. and Kenny, A.J. (1985) Biochem. J. 228, 119-126. LeBien, T.W. and McCormack, R.T. (1989) Blood 73, 625-635. Letarte, M., Vera, S., Tran, R., Addis, J.B.L., Onizuka, R.J., Quackenbush, E.J., Jongeneel, C.V. and McInnes, R.R. (1988) J. Exp. Med. 168, 1247-1253. M.A., Vijayaraghavan, J., Schmidt, E.V., Shipp, Masteller, E.L., D'Adamio, L., Hersh, L.B. and Reinherz, E.L. (1989) Proc. Natl. Acad. Sci. USA 86, 297-301. Malfroy, B., Kuang, W.J., Seeburg, P.H., Mason, A.J. and Schofield, P.R. (1988) FEBS Lett. 229, 206-210. Matthews, B.W., Colman, P.M., Jansonius, J.N., Titani, K., Walsh, K.A. and Neurath, H. (1972) Nature New Biol. 238, 41-43. Blumberg, S., Vogel, Z. and Altstein, M. (1981) Life Sci. 28, 301-306. Poszgay, M., Michaud, C., Liebman, M. and Orlowski, M. (1986) Biochemistry 25, 1292-1299. 625

Vol.

10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

172,

No.

2,

1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Erdas, E.G. and Skidgel, R.A. (1989) FASEB J. 3, 145-151. Zipori, D., Duskin, D., Tamir, M., Argaman, A., Toledo, J. and Malik, Z. (1985) J. Cell. Physiol. 122, 81-90. Benayahu, D., Kletter, Y., Zipori, D. and Wientroub, S. (1989) J. Cell. Physiol. 140, l-7. Indig, F.E., Ben-Meir, D., Spungin, A. and Blumberg, S. (1989) FEBS Lett. 255,237-240. Spungin, A. and Blumberg, S. (1989) Eur. J. Biochem. 183, 471-477. Fried, A., Benayahu, D., Zipori, D. and Wientroub, S. (1989) Calcif. Tissue Int. 44, (suppl.) S-48. Majeska, R.J., Rodan, S.B. and Rodan, G.A. (1980) Endocrinology 107, 1494-1503. Sudo, H., Kodama, H.-A., Amagai, Y., Yamamoto, S. and Kasai, S. (1983) J. Cell Biol. 96, 191-198. Robey, P.G. and Termine, J.D. (1985) Calcif. Tissue Int. 37, 453-460. Majeska, R.J. and Rodan, G.A. (1982) Calcif. Tissue Int. 34, 59-66. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. (1951) J. Lowry, O.H., Rosebrough, N.J., Fart-, A.H. and Randall, R.J. Biol. Chem. 193, 265-275. Benayahu, D., Fried, A., Zipori, D. and Wientroub, S. (1990) Calcif. Tissue Int. (accepted). Arock, M., Gros, C., Devillier, P., Guilloson, J.J. and Schwartz, J.C. (1989) FEBS Lett. 248, 123-126. Kenny, A.J., O'Hare, M.J. and Gusterson, B.A. (1989) Lancet, ii, 785787.

626