(Received for publication, August 17, 1994; and in revised form, October 28, 1994)
From the
Fibroblast growth factor (FGF)-1 is released from NIH 3T3 cells
in response to heat shock as a biologically inactive protein that is
unable to bind heparin and requires activation by
(NH)
SO
to generate a biologically
active extracellular heparin-binding growth factor (Jackson, A.,
Friedman, S., Zhan, X., Engleka, K. A., Forough, R., and Maciag,
T.(1992) Proc. Natl. Acad. Sci. USA 89, 10691-10695). To
further study the mechanism of FGF-1 release in response to heat shock
(42 °C), we examined the kinetics of FGF-1 release from
FGF-1-transfected NIH 3T3 cells and observed that the cells require at
least 1 h of exposure to heat shock conditions for the release of
FGF-1. Interestingly, agents that interfere with the function of the
endoplasmic reticulum-Golgi apparatus, exocytosis, and the multidrug
resistance pathway (brefelden A, methylamine, and verapamil,
respectively) do not inhibit the release of FGF-1 in response to
temperature; rather, they exaggerate the release of FGF-1. Because
immunoblot analysis of FGF-1 in the conditioned medium of heat-shocked
NIH 3T3 cells revealed the presence of a minor band with an apparent
molecular weight of a FGF-1 homodimer and because we have previously
shown that FGF-1, but not FGF-2, is able to form a homodimer in
response to chemical oxidation by CuCl
(Engleka, K. A., and
Maciag, T.(1992) J. Biol. Chem. 267, 11307-11315), we
examined whether reducing agents would substitute for
(NH
)
SO
and activate extracellular
FGF-1. Indeed, dithiothreitol and reduced glutathione are able to
individually generate a FGF-1 monomer as a heparin-binding protein from
the conditioned medium of heat-shocked NIH 3T3 cell transfectants. To
confirm that cysteine residues are involved in the release of FGF-1 in
response to temperature, we used mutagenesis to prepare a human FGF-1
Cys-free mutant in which Cys
, Cys
, and
Cys
were converted to serine. Analysis of the release of
the FGF-1 Cys-free mutant in NIH 3T3 cells transfected with the FGF-1
Cys-free mutant demonstrated that the FGF-1 Cys-free mutant is not
released into the conditioned medium in response to temperature.
Interestingly, exposure of the NIH 3T3 cell FGF-1 Cys-free
transfectants to brefelden A followed by heat shock also demonstrated
the absence of the extracellular FGF-1 Cys-free mutant. Finally,
ion-exchange and reverse-phase chromatographies of heat-shocked
conditioned medium analyzed by FGF-1 immunoblot analysis were able to
resolve FGF-1 as a homodimer under nonreducing conditions and as a
monomer under reducing conditions. These data demonstrate that FGF-1
utilizes cysteine residues as an important component of its release
from NIH 3T3 cells in vitro in response to temperature and
exits the cell as a biologically inactive homodimer with reduced
heparin affinity that requires activation by reducing agents to
generate heparin binding and biological activities.
The fibroblast growth factor (FGF) ()gene family is
presently composed of nine family members, and the prototype structures
for the FGF family are defined by FGF-1 (acidic) and FGF-2
(basic)(1) . A unique structural feature of the FGF prototypes
is the absence of classical signal peptide sequence to direct their
secretion through a conventional pathway utilizing the endoplasmic
reticulum (ER)-Golgi apparatus(1) . Because the biology of the
FGF prototypes is tightly coupled with developmental, neurotrophic, and
angiogenic processes and requires association with high affinity
cell-surface tyrosine kinase receptors to modify gene expression and
cell division(1) , it is important to understand the mechanism
by which the FGF prototypes are released from cells.
We have
previously shown that FGF-1 is released from FGF-1-transfected NIH 3T3
cells in response to heat shock in vitro in a form that is
biologically inactive and unable to associate with the
glycosylaminoglycan heparin(2) . However, the heparin binding
and growth promoting activities of FGF-1 released in response to
temperature can be recovered by (NH)
SO
precipitation of the conditioned medium(2) . While the
mechanism of FGF-1 release in response to temperature involves the
secretion of a latent protein, this mechanism appears to be specific
for FGF-1 since FGF-2 is not released from cells in vitro in
response to heat shock(3) . Thus, to further define the pathway
utilized by FGF-1 to gain access to the extracellular compartment in
response to temperature stress, we have studied the kinetics of FGF-1
release, the action of pharmacologic agents on the release of FGF-1,
and the ability of reducing agents to activate latent extracellular
FGF-1 in NIH 3T3 cells transfected with FGF-1. We report that the
pathway of FGF-1 release in response to heat shock involves the
function of cysteine residues that may be important for the secretion
of a latent FGF-1 homodimer.
The conditioned medium collected from
heat-shocked NIH 3T3 cell FGF-1 transfectants was also resolved by
Mono-S (Pharmacia Biotech Inc.) high pressure liquid chromatography
(HPLC). The medium (120 ml) was dialyzed against 50 mM sodium
phosphate (pH 6.0) containing 50 mM NaCl (20 h, 4 °C) and
fractionated with a linear gradient as described
previously(8) . Fractions (2 ml) containing FGF-1 were
collected, further resolved by Sep-Pak C HPLC,
lyophilized, resuspended in SDS-PAGE sample buffer, and analyzed by
immunoblot analysis using 12% (w/v) SDS-PAGE and FGF-1 antisera as
described above.
The RCPCR method was
also used to obtain a FGF-1 C30S mutant in pUC18. As described above,
four primers were used in two separate PCRs. One PCR was performed with
the primers AAGCCCAAACTCCTCTACTCTAGCAACGGG (sense oligonucleotide) and
CTTGTAATTCCCTGGAGGCAGATTAAA (antisense oligonucleotide), and the other
PCR was performed with the primers GAAGTGGCCCCCGTTGCTAGAGTAGAGGAG
(antisense oligonucleotide) and CTGAGGATCCTTCCGGATGGCACA (sense
oligonucleotide). The same four primers were also used for the
mutagenesis of Cys in the FGF-1 C131S mutant in pUC18. In
this case, the FGF-1 C30S/C131S mutant was obtained and eventually used
in the last set of RCPCRs for the mutagenesis of nucleotides encoding
Cys
in order to obtain the FGF-1 Cys-free mutant. In this
case, the primers CAGACACCAAATGAGGAATCTTTGTTCCTG (sense
oligonucleotide) and TGAGCCGTATAAAAGCCCGTCGGTGTC (antisense
oligonucleotide) were used in the first PCR, and the primers
CAGCCTTTCCAGGAACAAAGATTC (antisense oligonucleotide) and
GAGGAGAACCATTACAACACC (sense oligonucleotide) were used in the second
PCR. Sequence analysis demonstrated that two extra point mutations were
inadvertently introduced (TTC to TCC, F36S; GAA to GAT, E63D). To
obtain the FGF-1 Cys-free clone without the extra point mutations, the
FGF-1 C30S and FGF-1 C30S/F36S/E63D/C97S/C131S (five-point) mutants
were transferred into the prokaryotic expression vector pET3c. As
described previously (9) , PCR was used to add unique sites for
the restriction enzymes NdeI and BglII to the ends of
the DNA sequence encoding the two FGF-1 mutants. The FGF-1 C30S and
FGF-1 five-point mutants were amplified with 30 cycles using the
primers TACGGCATATGGCTGAAGGGGAAATCACC (sense oligonucleotide; NdeI site) and TACGAACAGATCTCTTTAATCAGAAGA (antisense
oligonucleotide; BglII site). The two NdeI- and BglII-digested inserts were subcloned into compatible NdeI/BamHI sites in the pET3c plasmid. The ligation
was performed with T4 ligase (Life Technologies, Inc.), and the
reaction products were confirmed by sequencing using the dideoxy method
with Sequenase (U. S. Biochemical Corp.) following the
manufacturer's instructions. The FGF-1 five-point pET3c plasmid
was then digested with NcoI and EcoRI; the band (741
base pairs) was purified and ligated into FGF-1 C30S pET3c previously
digested with NcoI and EcoRI, treated with calf
intestinal phosphatase, and gel band-purified prior to ligation. DNA
sequencing of the insert in pET3c confirmed the FGF-1 Cys-free
sequence, without the two extraneous mutations. The FGF-1 Cys-free
insert was transferred from pET3c into the eukaryotic expression vector
pMEXneo(4) . The FGF-1 Cys-free pET3c plasmid was digested with BamHI and EcoRI; the band (867 base pairs) was
purified and subcloned into BamHI/EcoRI-digested
pSF23
that had been treated with calf intestinal
phosphatase and purified by agarose electrophoresis prior to ligation.
DNA sequencing of the insert in pMEXneo confirmed the presence of the
FGF-1 Cys-free sequence.
The kinetics of FGF-1 release in response to temperature was
studied in an attempt to determine the rapidity of the release process in vitro. As shown in Fig. 1, we did not observe
significant levels of extracellular FGF-1 using
(NH)
SO
activation of the latent
heparin binding property of FGF-1 until after 1 h of exposure of the
FGF-1-transfected NIH 3T3 cell population to heat shock conditions.
While small levels of extracellular FGF-1 were present 30 min after
heat shock, we failed to detect extracellular FGF-1 at earlier time
points (Fig. 1A). In addition, we were unable to detect
S-labeled FGF-1 by pulse-chase analysis of
[
S]Met/Cys-labeled NIH 3T3 cell transfectants 60
min after heat shock using similar methods (data not shown). Because
the expression of FGF-1 in the NIH 3T3 cell transfectants is
constitutive and these cells contain cytosolic FGF-1 levels of
30
ng/10
cells, it was surprising that FGF-1 was released with
significantly delayed kinetics following heat shock. Thus, it is
possible that the cytosolic store of intracellular FGF-1 may not be
accessible to the temperature-induced FGF-1 release pathway, and this
is consistent with the identification of a cytosolic retention domain
in the structure of FGF-1. (
)
Figure 1:
A, kinetics of FGF-1 release in
response to temperature. Conditioned media were derived from NIH 3T3
cell pMEXneo/FGF-1 transfectants maintained either at 37 or 42 °C
for varied time periods as described previously(2) . The
conditioned media were collected, treated with 90% (w/v)
(NH)
SO
, centrifuged (9000
g, 40 min), resuspended in TEB, dialyzed against TEB (18 h, 4
°C), and adsorbed to heparin-Sepharose, and the 1.5 M NaCl
eluates were analyzed by immunoblot analysis for FGF-1. Lanes1 and 2 represent conditioned medium after 5 min
at 42 and 37 °C, respectively, and lanes3 and 4 (15 min), lanes5 and 6 (30 min), lanes7 and 8 (1 h), lanes9 and 10 (90 min), and lanes11 and 12 (2 h) represent conditioned medium harvested at the times
indicated in parentheses from control (37 °C; even-numbered
lanes) and heat-shocked (42 °C; odd-numbered lanes)
NIH 3T3 cells. B, activation of FGF-1 from heat-shocked
conditioned media using reducing agents rather than
(NH
)
SO
. Conditioned media were
derived from NIH 3T3 cell pMEXneo/FGF-1 transfectants maintained at 42
°C for 2 h. The conditioned media were collected and adsorbed to
heparin-Sepharose, and the 1.5 M NaCl eluates were analyzed by
immunoblot analysis for FGF-1. Lane1, conditioned
medium from heat-shocked (42 °C) cells; lane2,
conditioned medium from heat-shocked (42 °C) cells treated with
0.1% (w/v) DTT; lane3, conditioned medium from
heat-shocked (42 °C) cells treated with 1 mM glutathione
(oxidized form); lane4, conditioned medium from
heat-shocked (42 °C) cells treated with 1 mM glutathione
(reduced form). C, immunoblot analysis of FGF-1 from media
conditioned by heat shock and brefelden A. NIH 3T3 cell pMEXneo/FGF-1
transfectants were grown to confluence and pretreated with brefelden A
(0.5 µg/ml) for 30 min. Serum-free DMEM containing brefelden A (0.5
µg/ml) was added at time 0, and the monolayer was maintained at 42
°C for 2 h. Control NIH 3T3 cell pMEXneo/FGF-1 transfectants were
maintained under identical conditions at 37 and 42 °C. The
conditioned media were collected, 1 mM phenylmethysulfonyl
fluoride was added, the media were adsorbed with heparin-Sepharose, and
the 1.5 M NaCl eluates were analyzed by immunoblot analysis
for FGF-1 as described previously(2) . Lane1, conditioned medium from 37 °C control cells; lane2, conditioned medium from cells pretreated with
brefelden A at 37 °C; lane3, conditioned medium
from heat-shocked (42 °C) cells; lane4,
conditioned medium from heat-shocked (42 °C) cells pretreated with
brefelden A.
During the course of these (Fig. 1A, lanes7, 9, and 11) and prior (2) immunoblot experiments with
conditioned medium activated with
(NH)
SO
, we noted the appearance of
a minor band migrating with an apparent M
of
38,000, a position that corresponds with the apparent M
of a FGF-1 homodimer previously generated by
chemical oxidation of the recombinant human protein using
CuCl
(9) . Because the FGF-1 homodimer prepared by
chemical oxidation is biologically inactive and weakly associates with
heparin (9) and the form of FGF-1 released into the
conditioned medium in response to heat shock is also biologically
inactive and unable to associate with heparin at 0.7 M NaCl(2) , we questioned whether we could recover the
heparin binding properties of FGF-1 from the heat-shocked conditioned
medium using reducing agents rather than by activation with
(NH
)
SO
. As shown in Fig. 1B, 0.1% (w/v) dithiothreitol (DTT) and 1 mM reduced glutathione, but not 1 mM oxidized glutathione,
were able to activate the heparin binding property of FGF-1 in the
conditioned medium of heat-shocked NIH 3T3 cell FGF-1 transfectants.
These data suggest that FGF-1 may be released into the extracellular
compartment as a latent FGF-1 complex that may be associated with
itself or another protein by a disulfide bridge since reducing agents
such as DTT and glutathione are able to substitute for
(NH
)
SO
and activate the heparin
binding activity of FGF-1 released into the extracellular compartment
in response to temperature stress.
We also utilized pharmacologic agents known to impair the secretory function of the ER-Golgi apparatus(11) , exocytosis(12) , and the multidrug resistance pathway (13) in order to determine whether these pathways are utilized during FGF-1 release in response to temperature stress. As shown in Fig. 1C, NIH 3T3 cells transfected with FGF-1 released FGF-1 into the conditioned medium as a result of heat shock for 2 h at 42 °C. However, rather than inhibit the release of FGF-1 in response to temperature, pretreatment of the NIH 3T3 cell transfectants with brefelden A (0.5 µg/ml) resulted in an exaggerated level of FGF-1 in the conditioned medium (Fig. 1C). Similar results were also obtained by pretreatment of the FGF-1-transfected NIH 3T3 cell monolayer for 30 min with 10 mM methylamine and 10 µg/ml verapamil (data not shown). These results suggest that the release of FGF-1 in response to temperature utilizes a pathway that is independent of the conventional secretory pathway mediated by the ER-Golgi apparatus.
To further
define the form of FGF-1 released by NIH 3T3 cell FGF-1 transfectants
in response to temperature stress, FGF-1 present in the conditioned
medium was fractionated by ion-exchange HPLC (Fig. 2A).
FGF-1 immunoblot analysis of this sample performed in the absence of
reducing agents resolved a series of bands with apparent M values between 33,000 and 36,000, while
identical analysis in the presence of a reducing agent revealed a
single band with an apparent M
of 17,000 (Fig. 2B). The triplet band observed in the nonreduced
sample was similar to that previously observed with the FGF-1 homodimer
prepared by chemical oxidation with CuCl
(9) .
Indeed, the recombinant FGF-1 standard also contained a small level of
the triplet FGF-1 homodimer when the protein was resolved in the
absence of reductant (Fig. 2B, thirdlane). These data suggest that FGF-1 is released into the
conditioned medium in response to temperature as a homodimer.
Figure 2: A, ion-exchange HPLC of FGF-1 released in response to heat shock. NIH 3T3 cell FGF-1 transfectants were exposed to temperature stress, and conditioned media were collected and processed as described under ``Methods and Materials.'' The post-Mono-S fraction defined by the boundary was pooled, adsorbed to a Sep-Pak resin (Waters), and eluted with 50% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid. The fraction was lyophilized and processed for FGF-1 immunoblot analysis. B, immunoblot analysis of the fractions collected by ion-exchange HPLC. The fractions were resuspended in SDS-PAGE sample buffer with or without DTT and resolved by SDS-PAGE, and FGF-1 was identified as described under ``Methods and Materials.'' Lane3 contains recombinant human FGF-1 without DTT.
To
confirm the importance of cysteine residues for the release of latent
FGF-1 in response to heat shock, we utilized the recombinant circle
polymerase chain reaction strategy to prepare a human FGF-1 Cys-free
mutant. Expression of the FGF-1 Cys-free mutant in the prokaryotic
pET3c expression system yields a recombinant protein with heparin
binding properties and mitogenic activity similar to those previously
reported (8) for wild-type recombinant human FGF-1 (data not
shown). This is consistent with the report that cysteine residues are
not required for biological activity(14) . NIH 3T3 cells were
transfected with the FGF-1 Cys-free mutant using the eukaryotic
expression vector pMEXneo(4) , and the release of the FGF-1
Cys-free mutant in response to heat shock was examined using the NIH
3T3 cell transfectants. As shown in Fig. 3A, we were
readily able to detect the FGF-1 Cys-free mutant in the cytosol of
heat-shocked NIH 3T3 cell transfectants. However, we were unable to
detect the FGF-1 Cys-free mutant in the extracellular compartment
following (NH)
SO
activation of the
conditioned medium (Fig. 3A). Because brefelden A
pretreatment of NIH 3T3 cell FGF-1 transfectants resulted in an
enhanced level of FGF-1 in the conditioned medium after heat shock (Fig. 1C), we examined whether the FGF-1 Cys-free
mutant could be released into the conditioned medium following heat
shock of the NIH 3T3 cell FGF-1 Cys-free mutant transfectants
pretreated with brefelden A. As shown in Fig. 3B, we
were unable to detect the appearance of the FGF-1 Cys-free mutant in
the conditioned medium of the brefelden A-pretreated NIH 3T3 cell FGF-1
Cys-free mutant transfectants following
(NH
)
SO
activation of the
conditioned medium. Additional experiments using 0.1% (w/v) DTT
activation yielded results similar to those shown in Fig. 3(A and B). These results suggest that
the release of FGF-1 in response to temperature involves the
intracellular function of FGF-1 cysteine residues that may play a role
in the formation of a latent homodimer.
Figure 3:
Release of FGF-1 Cys-free mutant in
response to brefelden A and heat shock. NIH 3T3 cells were transfected
with the FGF-1 Cys-free mutant as described under ``Materials and
Methods.'' A, immunoblot analysis of FGF-1 and FGF-1
Cys-free release in response to heat shock. FGF-1-transfected (30
ng of intracellular FGF-1/10
cells) and FGF-1 Cys-free
mutant-transfected (
90 ng of intracellular FGF-1/10
cells) NIH 3T3 cells were subjected to heat shock (2 h, 42
°C); conditioned media were collected, treated with 90% (w/v)
(NH
)
SO
, processed as described in
the legend to Fig. 1, and adsorbed to heparin-Sepharose; and the
1.5 M NaCl eluates were analyzed by immunoblot analysis for
FGF-1. Lane1, conditioned medium from control (37
°C) FGF-1 Cys-free transfectants; lane2,
conditioned medium from heat-shocked (42 °C) FGF-1 Cys-free
transfectants; lane3, conditioned medium from
control (37 °C) FGF-1 transfectants; lane4,
conditioned medium from heat-shocked (42 °C) FGF-1 transfectants; lane5, cell lysate from FGF-1 Cys-free
transfectants; lane6, cell lysate from control FGF-1
transfectants. B, immunoblot analysis of FGF-1 and FGF-1
Cys-free release in response to brefelden A pretreatment and heat
shock. FGF-1-transfected and FGF-1 Cys-free mutant-transfected NIH 3T3
cells were treated with brefelden A as described under ``Materials
and Methods'' and subjected to heat shock (2 h, 42 °C);
conditioned media were collected, treated with
(NH
)
SO
as described above, and
adsorbed to heparin-Sepharose; and the 1.5 M NaCl eluates were
analyzed by immunoblot analysis for FGF-1. Lane1,
conditioned medium from FGF-1 Cys-free transfectants pretreated with
brefelden A; lane2, conditioned medium from control
FGF-1 Cys-free transfectants; lane3, control FGF-1
transfectants pretreated with brefelden A; lane4,
control FGF-1 transfectants.
The secretory pathway
utilized by FGF-1 is unique since FGF-2 is not released from NIH 3T3
cells transfected with FGF-2 in response to heat shock (3) and,
unlike FGF-1, FGF-2 does not readily associate to form FGF-2 homodimers
in response to Cu oxidation (9) even though 2
of the 3 cysteine residues are conserved between FGF-1 and
FGF-2(1) . However, we have not eliminated the possibility that
FGF-1
FGF-2 heterodimers may be released from NIH 3T3 cells in
response to temperature. Thus, our data suggest that in response to
temperature stress, FGF-1 is released into the extracellular
compartment as a functionally inactive homodimer that is able to
associate poorly with immobilized heparin, and the latent FGF-1
homodimer may be activated by reducing agents such as glutathione to
generate heparin binding and mitogenic activities. Furthermore, this
mechanism argues that the redox state within tissue microenvironments
may play a major role in the pathway of hypoxia-mediated angiogenesis
and may also resolve the functional importance of adding reducing
agents to cell culture environments for the maximal growth stimulation
of a variety of diploid mammalian cell types.