(Received for publication, October 21, 1994; and in revised form, May 12, 1995)
From the
Mounting experimental evidence suggests that the TAT protein,
released from human immunodeficiency virus-1 (HIV-1)-infected
inflammatory cells, may genetically reprogram targeted cells within a
localized environment to develop highly vascularized tumors of
mesenchymal origin. The fibroblast growth factor (FGF) family of
polypeptides has gained general acceptance as initiators of
angiogenesis and functions as potent mitogens for mesoderm-derived
cells. To evaluate a potential biological relationship between TAT and
acidic FGF (FGF-1), primary murine embryonic fibroblasts either were
transfected with the viral transactivator or were transduced
(retrovirally mediated) with a secreted, chimeric form of the human
polypeptide growth factor, human stomach tumor/Kaposi's sarcoma
(hst/KS)FGF-1. Reverse transcriptase-polymerase chain reaction, Western
blotting, in situ immunohistochemical, heparin affinity, DNA
synthesis, and transient transfection techniques were used to confirm
expression, localization, and functionality of the transgenes. Both
transfected and transduced cells constitutively expressing either TAT
or (hst/KS)FGF-1 adopted a transformed phenotype, maintained aggressive
growth behavior, and demonstrated both induction of FGF-specific
phosphotyrosyl proteins and nuclear association of FGF-1 and FGF-1
receptor. Increased levels of endogenous, murine FGF-1 mRNA (reverse
transcriptase-polymerase chain reaction) and protein (immunoblot
analysis) were apparent in both (hst/KS)FGF-1- and TAT-transformed
cells. Medium conditioned by (hst/KS)FGF-1-transduced cells contained
steady-state levels of biologically active FGF-1 which exhibited a
representative molecular weight. Limited sodium dodecyl
sulfate-polyacrylamide gel electrophoretic analysis of the conditioned
medium from TAT-transformed cells demonstrated the appearance of FGF-1
as latent, high molecular weight complexes requiring reducing agents to
activate full biological activity. Collectively, these results suggest
that TAT induces the expression and secretion of FGF-1, which may be
potentially relevant to the pathophysiological development of
AIDS-Kaposi's sarcoma.
Although the human immunodeficiency virus-1 (HIV-1)
The extracellular appearance of FGF is particularly relevant
since this family of polypeptides serves as potent mitogens for
mesoderm-derived cells in vitro and functions as hormone
inducers of angiogenesis in vivo(26, 27) .
Interestingly, FGF-1 and FGF-2 are unusual in that, unlike the other
seven FGF family members, they lack a classical signal sequence for
secretion, a unique feature also shared by IL-1
Under normal
physiologic conditions, intracellularly sequestered FGF may not be
available to mediate its full biological potential.
Oxidative stress is recognized to be associated with most
pathophysiological conditions, including HIV-1 infection(49) .
HeLa cells stably transfected with the TAT gene have demonstrated
evidence of increased oxidative stress by suppressing expression of
manganese superoxide dismutase(15) , an observation reported
previously in cells acutely infected with HIV-1(49) .
Attenuation of manganese superoxide dismutase results in accumulation
of reactive oxygen species, which has been demonstrated to induce
cellular proliferation(15, 50) . Since oxidative
stress, heat shock, and serum starvation responses overlap (51, 52, 53, 54) , the role of TAT
as a stress inducer for the secretion of FGF-1 was examined in
vitro. Several lines of evidence provide a compelling argument
that TAT-induced cellular growth may include not only increased
synthesis of FGF-1 but also secretion of this polypeptide as a high
molecular weight, noncovalent complex, an observation that may be
relevant to the development and progression of AIDS-KS.
Figure 1:
Structure
of the eukaryotic expression vectors. The expression vector pMEXneo
containing the murine sarcoma virus (MSV) LTR promoter and
SV40 polyadenylation site (pA) was utilized for the expression
of the HIV-1 TAT protein following subcloning into the multiple cloning
site (MCS). Each of the retroviral vector constructs contains
a 5`-LTR directing expression of
Total recovered proteins
from individual cell populations were processed separately by affinity
extraction. To obtain TAT, proteins processed with ammonium sulfate
were resuspended (GuHCl) and loaded on a chromatographic cartridge
packed with a reversed phase C18 resin (Sep-Pak, Rainin) and
equilibrated in 0.1% (v/v) trifluoroacetic acid. Bound proteins were
washed (0.1% trifluoroacetic acid), eluted (40% acetonitrile, 0.1%
trifluoroacetic acid), lyophilized (Speed-Vac), and resuspended with
0.5 M DTT in TE. To extract native FGF-1, recovered proteins,
processed either with or without ammonium sulfate, were resuspended
(0.5 M NaCl in TE) and loaded on a heparin-Sepharose column
(equilibrated in resuspension buffer) using a fast protein liquid
chromatography system (Pharmacia Biotech Inc.). To extract total
extracellular heparin-binding proteins, including complexed forms of
FGF-1, processed conditioned medium was loaded on a heparin-Sepharose
column equilibrated in TE. Heparin-binding proteins were eluted (2.0 M NaCl in TE) and concentrated/desalted with a Centricon-3
(Amicon).
Immunohistochemical localization of TAT, FGF-1, and FGFR-1 was
performed using Vectastain Elite ABC following the manufacturer's
recommendations (Vector Laboratories). Cells were centrifuged, fixed,
and depleted of endogenous peroxidase activity as described above.
Fixed cells were washed (TTBS) and incubated (30 min) with either
anti-TAT (AL72, 1:100), affinity-purified anti-FGF-1 (1.5 µg/ml),
or affinity-purified anti-FGFR-1 (5 µg/ml). Negative controls
included incubation of fixed cells with preimmune rabbit serum (10%)
and deletion of the primary antibody. An additional control for TAT and
FGF-1 specificity included antibody blocking conditions as described
for Western analyses, which completely abrogated cellular staining.
Antibody-treated samples were incubated with biotinylated secondary
antibody (goat, anti-rabbit) followed by avidin-biotin horseradish
peroxidase (Vector ABC). Chromogenic development of stained samples (as
described above) permitted analysis via light microscopy.
Primary cultures of embryonic fibroblasts were transfected or
transduced independently with expression vectors (Fig. 1),
selected in Geneticin or by FACS, and expanded for biochemical
analyses. The transduction efficiency, as determined by percentage of
cells expressing
Figure 2:
Steady-state levels of mRNA in individual
cell populations. Total RNA (1.0 µg), isolated from separate
confluent monolayers of different cell populations, was used in reverse
transcriptase-PCR assays (35 cycles) with specific sense and antisense
primers. The amplification products were separated on a 2% (w/v)
agarose gel and stained with ethidium bromide. Each gel contained a
1.0-kilobase pair DNA ladder (Life Technologies, Inc.) which served as
a molecular mass marker (lanes 1 and 10). Panel
A, reverse transcriptase-PCR analysis of transfected cell
populations. Lanes 2, 4, and 6, pMN RNA; lanes 3, 5, and 7, pTAT RNA; lanes 6 and 7, reverse transcriptase-PCR without reverse
transcriptase; lanes 8 and 9, PCR analysis of pTAT
plasmid DNA (2.0 ng). Amplimer pairs used to analyze individual
transfectants and controls were: N5/N3, lanes 2, 3, 6, 7, and 8; T5/T3, lanes 4, 5, and 9. Panel B, reverse transcriptase-PCR
analysis of transduced cell populations. Lanes 2, 4,
and 6, Bg RNA; lanes 3, 5, and 7,
(hst/KS)FGF-1 RNA; lanes 6 and 7, reverse
transcriptase-PCR without reverse transcriptase; lanes 8 and 9, PCR analysis of (hst/KS)FGF-1 plasmid DNA (2.0 ng).
Amplimer pairs used to analyze individual transductants and controls
were: B5/B3, lanes 2, 3, 6, 7, and 8; F5/F3, lanes 4, 5, and 9. Panel C, reverse transcriptase-PCR analysis of full-length,
endogenous murine FGF-1 in individual cell populations. Specific sense
and antisense primers are described under ``Materials and
Methods.'' Lane 2, pMN RNA; lane 3, pTAT RNA; lane 4, Bg RNA; lane 5, (hst/KS)FGF-1 RNA; lane
6, PCR analysis of plasmid DNA (2.0 ng) containing full-length
FGF-1
Figure 3:
Western analyses of individual cell
populations. Panel A, extracted proteins harvested from both
total cellular (1
The morphological
differences between individual populations of transfected/transduced
cells were compared microscopically. Control cells transfected with pMN (Fig. 4A) or transduced with Bg (Fig. 5A) displayed a typical monolayer phenotype. In
contrast, cells expressing TAT (Fig. 4B) or
(hst/KS)FGF-1 (Fig. 5B) demonstrated a spindle-like
morphology characterized by exaggerated foci formation at a high
density with increased cell motility and a general loss of anchorage
dependence for growth. Nontransfected/transduced primary murine
fibroblasts adopted this characteristic transformed phenotype when
treated daily for 3 days with recombinant FGF-1 (50 ng/ml) in
combination with heparin (20 USP units/ml). The morphological response
to these daily treatments was dependent on constant presentation of the
growth factor since its elimination resulted in reversion to a normal
phenotype.
Figure 4:
In situ analyses of individual
transfected cell populations. The phenotypes of pMN- (panel A)
and pTAT-(panel B) transfected cells (9
Figure 5:
In situ analyses of individual
transduced cell populations. The phenotypes of Bg- (panel A)
and (hst/KS)FGF-1- (panel B) transduced cells (9
Figure 6:
Growth behavior of transfected and
transduced cells. Individual cell populations were grown in duplicate
as described under ``Materials and Methods'' in the presence
of either 10% v/v (panels A and C) or 0.5% v/v (panels B and D) FBS. The kinetics of growth behavior
were determined for cells either transfected (panels A and B) with pMN (
Figure 7:
In situ DNA synthesis assay. Panel A, proliferation index of individual cell populations.
Subconfluent populations of cells (4
In situ immunohistochemical methods were used to
confirm Western analyses of individual cell populations ( Fig. 4and Fig. 5). In contrast to pMN transfectants (Fig. 4C), cells transfected with pTAT exhibited
immunoreactivity for TAT not only distributed within the cytosol but
also localized predominantly in the nuclear compartment of greater than
95% of the cell population (Fig. 4D). Both pMN
transfectants (Fig. 4, E and G) and Bg
transductants (Fig. 5, C and E) displayed
immunoreactivity for FGF-1 and FGFR-1 which was distributed largely
within the cytoplasmic compartment with occasional arrays confined to
the plasma membrane. In contrast, both pTAT (Fig. 4, F and H) and (hst/KS)FGF-1 (Fig. 5, D and F) cells exhibited immunostaining for the growth factor and
receptor which was associated predominantly with the nuclear
compartment in greater than 95% of the total cell population.
To
correlate nuclear immunoreactivity with the extracellular compartment,
conditioned media from pMN- and pTAT-transfected cells were examined
for the presence of FGF-1. Immunoblot analysis of proteins exhibiting
heparin affinity in 0.5 M NaCl failed to detect soluble FGF-1
in media conditioned by either transfectant (Fig. 3C, lane 4). To examine the possibility that extracellular FGF-1
existed as a complex with reduced heparin affinity, conditioned media
from individual cell transfectants were isolated initially by ammonium
sulfate precipitation (90% saturation) followed by heparin extraction.
Western analysis of processed conditioned media demonstrated that the
extracellular appearance (approximately 5 ng/10
The truncated human
FGF-1 transgene, affinity extracted from the total intracellular or
extracellular compartment of (hst/KS)FGF-1-transduced cells, displayed
mitogenic behavior in a DNA synthesis assay similar to that obtained
with recombinant human FGF-1
To validate further the
biological potential of extracellular FGF-1, endogenous levels of
tyrosine phosphorylation were examined in individual cell populations.
When compared with quiescent pMN cells (Fig. 8A, lane 1), pTAT transfectants (Fig. 8A, lane
5) demonstrated increased tyrosine phosphorylation. Also, enhanced
phosphorylation of endogenous cortactin, migrating with an apparent
molecular mass of approximately 80 kDa, was evident in pTAT cells (Fig. 8B). Increased tyrosine phosphorylation of
endogenous c-src, migrating with an apparent molecular mass of
60 kDa, also was observed in pTAT-transfected cells (data not shown).
When compared with quiescent Bg cells (Fig. 8C, lane 1), (hst/KS)FGF-1-transduced cells also demonstrated
exaggerated tyrosine phosphorylation of specific polypeptides (Fig. 8C, lane 2), including endogenous
cortactin (Fig. 8D) and c-src (data not
shown). Medium conditioned by pTAT cells induced similar increases in
tyrosine phosphorylation (Fig. 8A, lane 2), an
observation that was exaggerated by pretreatment of the conditioned
medium with DTT (Fig. 8A, lane 3).
Phosphorylation of tyrosine residues on polypeptides with apparent
molecular masses of approximately 150, 130, and 90 kDa was consistent
with those substrates responding to exogenous recombinant FGF-1 (Fig. 8, panel A, lane 4; panel C, lane 3), FGF-1 extracted from medium conditioned by
(hst/KS)FGF-1 cells (Fig. 8C, lane 4), or
unprocessed medium conditioned by (hst/KS)FGF-1 cells (Fig. 8C, lane 5). Collectively, these results
correlate with the requirement for reducing agents to activate the
mitogenic potential of latent, extracellular FGF-1 released from
TAT-expressing cells.
Figure 8:
Autocrine/paracrine stimulation of
tyrosine phosphorylation. Individual cell extracts (1
An aggressive form of KS associated with HIV-1 infection is
classified as a highly vascularized tumor of mesenchymal origin
characterized by transformed spindle-shaped cells of unknown ontogeny (21, 22, 70) . These lesions are maintained
within a neovascular stroma of granulation tissue containing recruited
fibroblast, inflammatory, and endothelial cells(71) . A direct
involvement of HIV in KS development is unlikely since genomic
sequences of the virus have not been detected in KS
biopsies(72, 73) . Experimental evidence indicates
that the product of the HIV-1 TAT gene is released by acutely infected
T cells(20, 74) . Conditioned medium from these cells,
as well as recombinant TAT, has been shown to promote the growth of
AIDS-KS spindle cells in vitro(20, 74) , a
process that is inhibited by the inclusion of antibodies specific for
TAT(74) . Therefore, TAT may trigger a cascade of events in
responding cells which represents a biological link between HIV
infection and the development of AIDS-KS.
Previous in vitro studies with established cell lines have demonstrated that
presentation or expression of TAT induces cellular growth, a
transformed phenotype, and modulation of several endogenous
genes(75) . In studies reported here, expanded cultures of
primary fibroblasts, transfected with TAT, exhibited a transformed
phenotype characterized by: (i) altered morphology; (ii) loss of
contact inhibition; (iii) anchorage-independent growth; and (iv)
enhanced growth potential. Readily detectable levels of TAT mRNA were
translated into biologically active protein, including steady-state
levels of immunoreactive polypeptide associated with both the nuclear
and extracellular compartments. Collectively, these observations
provide a first in vitro indication in primary cells that
constitutive expression of TAT is capable of inducing cellular
transformation.
Several results obtained from the experimental
models described here provide insight into the pathophysiological
consequences of intrinsic TAT expression beyond that reported
previously. Compared with controls, TAT-transfected cells demonstrated
(i) increased expression of FGF-1 mRNA and protein; (ii) the appearance
of immunoreactive FGF-1 and FGFR-1 associated with the nuclear
compartment; (iii) retention of a high proliferation index that was
decreased by the anti-FGF-1 antibody; (iv) an increase of endogenous
FGF-specific tyrosine phosphorylation; and (v) the ability to condition
medium capable of stimulating tyrosine phosphorylation of FGF-specific
polypeptide substrates. Collectively, these results predict the
involvement of FGF-1 in TAT-induced growth.
The mitogenic activity
of FGF-1 involves nuclear association of the polypeptide by an
extracellular pathway which can occur either through high affinity
receptors and associated signal transduction events (37, 38, 39) or by transport of the
polypeptide into the cell in the absence of tyrosine
phosphorylation(76) . Results here are consistent with the
former mechanism, wherein constitutive expression of TAT-induced
nuclear association of FGFR-1 and FGF-1 as well as FGF-specific
tyrosine phosphorylation, including that associated with both the
FGFR-1 substrate, c-src(77) and cortactin, a
substrate for c-src which responds to extracellular FGF-1 (78) . In fact, intracellularly sequestered FGF-1 may not be
available to mediate its effects on cellular proliferation. Primary
murine fibroblasts constitutively expressing a 10-20 fold
increase of active FGF-1 under normal culture conditions maintain: (i)
a typical cellular phenotype; (ii) contact inhibition at high density;
(iii) anchorage-dependent growth; (iv) a low proliferation index; (v)
the absence of FGF-specific hyperphosphotyrosyl proteins; and (vi) the
absence of nuclear localized FGF-1.
Since nuclear localization of FGF-1 and
FGFR-1 requires an extracellular
pathway(40, 41, 42) , it seemed reasonable to
anticipate the appearance of FGF-1 in medium conditioned by
TAT-transfected cells. The ability of the antibody against FGF-1 to
decrease the growth of TAT-transfected cells is consistent with this
hypothesis. Interestingly, immunoblot detection of the secreted form of
FGF-1 in response to TAT expression required treatment with ammonium
sulfate to activate heparin binding, an observation consistent with
temperature-induced secretion of the growth
factor(47, 48) . Additional efforts demonstrated
further that FGF-1 is released from TAT-transfected cells as
noncovalent complexes, which exhibit decreased affinity to immobilized
heparin and resolve as multiple high molecular mass bands following
nondenaturing limited SDS-PAGE. Although the exact identity of the
extracellular, high molecular mass FGF-1 complexes are not known, our
data are consistent with recent observations that FGF-1 is released in
response to heat shock (47) and serum deprivation
In additional
studies reported here, expanded cultures of primary murine
fibroblasts, transduced with the (hst/ KS)FGF-1 chimera, also
demonstrated a transformed phenotype, characterized by (i) altered
morphology; (ii) lack of contact inhibition; (iii)
anchorage-independent growth; (iv) enhanced growth behavior; and (v) a
high proliferation index. Furthermore, readily detectable levels of
(hst/KS)FGF-1 mRNA were translated into active protein, including
steady-state levels of a soluble, extracellular form that retained: (i)
a representative molecular mass; (ii) characteristic affinity to
immobilized heparin; (iii) immunoreactivity to anti-FGF-1 antibodies;
(iv) mitogenic activity; and (v) the ability to induce FGF-specific
tyrosine phosphorylation. Compared with controls and similar to TAT
transfectants, (hst/KS)FGF-1-transduced cells further demonstrated: (i)
the appearance of immunoreactive FGF-1 and FGFR-1 associated with the
nuclear compartment; (ii) retention of a high proliferation index that
was decreased by the anti-FGF-1 antibody; (iii) an increase of
endogenous FGF-specific tyrosine phosphorylation; and (iv) the ability
to condition media competent to paracrine-stimulate FGF-specific
tyrosine phosphorylation. Collectively, these observations are
consistent with previous studies (34, 35, 36) and confirm the ability of
FGF-1, secreted as a native structure, to transform primary murine
fibroblasts as defined by specific biological responses, each of which
can be documented in TAT-transformed cells. In addition,
(hst/KS)FGF-1-transduced cells demonstrated increased levels of
endogenous, full-length murine FGF-1 mRNA and protein, which is
consistent with the potential of extracellular FGF-1 to
transcriptionally regulate its own promoter(81) . This feature
not only supports the potential existence of extracellular FGF-1 in
TAT-transfected cells but also suggests a mechanism whereby TAT may
induce the expression of FGF-1. Alternatively, TAT may, in a manner
similar to its role in the HIV-1 life cycle(82) , exert its
transcription effects directly on native FGF-1 production, which has
been demonstrated to experience poor expression as a consequence of
potential mRNA stem loop structures(57) . More rigorous
mechanistic studies, however, will be required to elucidate the
molecular events regulating TAT-induced expression of FGF-1.
Therefore, primary murine fibroblasts expressing TAT not only
exhibit aberrant phenotypic appearance and growth behavior but also
demonstrate the extracellular appearance of FGF-1 which, in its native
form, is a competent transforming polypeptide. To conclude that an
FGF-1 autocrine/paracrine loop is responsible for TAT-induced growth
and transformation is limited by the fact that (i) the detectable,
secreted FGF-1 complexes are biologically inactive, and (ii) numerous
other eukaryotic genes with established transforming potential are
induced by TAT. However, results presented here do suggest that
TAT-transfected cells maintain an intrinsic ability to activate some of
the latent, extracellular FGF-1 complexes, thereby permitting the
observed receptor-mediated mitogenic responses. The appearance of
characteristics associated with FGF-1-induced growth (including nuclear
association of ligand and receptor, decreased mitogenic behavior in
response to extracellular antibodies against FGF-1, etc.) and the
absence of steady-state levels of detectable FGF-1 as a native
structure would be consistent with this interpretation. Although the
exact biological mechanism responsible for activating the extrinsic
FGF-1 complex is not known, we suggest that an increased extracellular
reductive potential may be involved. The ability of reducing agents to
dissociate the secreted FGF-1 complexes, activate the full mitogenic
potential of extracellular FGF-1, and exaggerate paracrine stimulation
of FGF-specific tyrosine phosphorylation is consistent with this
hypothesis. Secretion of increased redox potential, which has
accumulated as a consequence of maximum oxygen utilization and nutrient
consumption(83) , is anticipated, particularly in
TAT-transfected cells experiencing rapid growth in vitro.
Whether this process provides sufficient levels of activated FGF-1 to
sustain transformation remains unclear and will require additional
studies.
Whereas the mechanism of FGF-1 release in response to TAT
expression involves the secretion of a latent protein complex, the
process by which this occurs remains to be elucidated. Potential FGF-1
homodimer formation argues that the intracellular redox state may play
a role in this pathway. Oxidative stress is recognized to be associated
with most pathophysiological conditions, including HIV-1
infection(49) . In addition, HeLa cells stably transfected with
the TAT gene demonstrated evidence of increased oxidative stress by
suppressing expression of manganese superoxide dismutase(15) ,
an observation reported previously in cells acutely infected with
HIV(49) . Attenuation of manganese superoxide dismutase would
result in increased levels of reactive oxygen species, which have been
demonstrated to induce both cellular proliferation (15, 50) and expression of
c-myc(84) , an established FGF response
gene(27) . Since (i) c-myc is capable of inducing
expression of heat shock proteins(43) , (ii) oxidative stress
and heat shock responses overlap(51) , and (iii) hyperthermic
stress induces the secretion of FGF-1(44, 48) , it is
suggested that TAT induces the extracellular appearance of FGF-1 by an
indirect mechanism involving biological stress. Whether TAT-induced,
FGF-1 secretion functions in vivo to initiate, coordinate, and
regulate the cellular proliferation associated with the
pathophysiological development of AIDS-KS remains the focus of
additional efforts.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U26456[GenBank® Link].
We thank S. Nan, M. L. Spell, J. Jin, X. Liu, J. Crow,
and Q. Li for excellent technical assistance; B. Johnson for expert
secretarial assistance; Alan Frankel, Whitehead Institute, for
generously providing polyclonal AL72 and E. coli expressing
recombinant TAT; Gilbert Jay, American Red Cross, for the pHIV/LTR-tat3
construct; Robert Friesel, American Red Cross, for the polyclonal
antibodies against FGFR-1 and phosphotyrosine; Xi Zhan, American Red
Cross, for the polyclonal antibody against cortactin; and Lisle Nabell,
University of Alabama at Birmingham, for the HIV-LTR-luciferase
reporter plasmid.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)genome contains a number of regulatory genes,
special attention has been given to understanding the biology of TAT, a
potent transactivator of viral gene transcription (1, 2, 3, 4) . The introduction of
the TAT gene into the germ line of mice under the control of the HIV
LTR (5) has yielded the appearance of dermal lesions that
histologically resemble Kaposi's sarcoma (KS). An implication of
this transgenic data is that TAT may act as a selective transcriptional
regulator of eukaryotic genes that function to initiate and sustain KS.
Indeed, numerous in vitro studies have demonstrated TAT to
modulate expression of eukaryotic genes, including transforming growth
factor-
(6) and
-
(7, 8, 9) , extracellular matrix
proteins(10) , IL-2(11, 12) ,
IL-6(13, 14) , manganese superoxide
dismutase(15) , and receptors for tumor necrosis factor-
(16) , IL-2(11, 17) , and IL-4(18) .
In addition, following absorptive endocytotic uptake (19) and
prior to nuclear translocation(20) , extracellular TAT can
stimulate the growth of cells(9, 17) , including those
derived from human AIDS-KS lesions(20, 21) .
Collectively, these in vitro observations support the
suggestion that HIV-1-infected inflammatory cells may release a
polypeptide mitogen (TAT) in vivo which initiates a localized
biological cascade of molecular events resulting in the subsequent
production of growth factors and cytokines relevant to the initiation
and perpetuation of proliferating KS
lesions(21, 22, 23, 24) . Indeed,
cells derived from AIDS-KS demonstrate increased levels of mRNAs
encoding several growth promoting factors, including acidic and basic
fibroblast growth factor (FGF-1 and FGF-2, respectively), IL-1 (
and
), transforming growth factor-
, platelet-derived growth
factor, and granulocyte-macrophage colony-stimulating
factor(25) . In addition, immunoprecipitation studies have
detected the appearance of biologically active IL-1
and FGF-2 in
medium conditioned by AIDS-KS cells in vitro(25) .
However, mechanisms whereby TAT transactivates expression and
extracellular presentation of these factors have not been elucidated
fully.
and
TAT(28, 29) . Transfection studies with FGF chimeras
containing various signal sequences(30, 31, 32, 33, 34, 35, 36) have
described the ability of secreted FGF-1 and FGF-2 to mediate cellular
transformation. These and other studies have established that the
biological activity of FGF-1 involves productive interactions with
specific high affinity receptors on the cell surface (37, 38, 39) followed by activation of
intrinsic tyrosine kinase(37, 38) , phosphorylation of
specific polypeptides(37) , nuclear translocation of the
ligand(40, 41) , and association of the high affinity
receptor (FGFR-1) with the nuclear compartment as a structurally intact
and functional tyrosine kinase(42) .
(
)Consequently, mechanisms whereby the cytosolic
growth factors are released into the extracellular environmment become
fundamental for understanding the regulation of FGF biology. In the
absence of defined mechanisms regulating the release of FGF prototypes
from intracellular stores, most studies have proposed that the
extracellular appearance of FGF-1 and FGF-2 is the consequence of cell
death/damage(44, 45, 46) . However, previous
results have determined that FGF-2 can be secreted from cytoplasmic
stores by an exocytosis pathway independent of the endoplasmic
reticulum-Golgi apparatus(44) . In addition, FGF-1 has been
demonstrated to be released from NIH 3T3 cells in response to heat
shock (47) by an endoplasmic reticulum-independent pathway as a
latent complex, which requires activation by reducing
agents(48) . More recent efforts
have suggested
that primary murine embryonic fibroblasts, transduced with the native
human FGF-1 gene, are able to secrete this growth factor in response to
an altered redox potential associated with serum starvation.
Recombinant Plasmids
To construct the
plasmid pTAT (Fig. 1), the NarI/SalI fragment
from pHIV/LTR-tat3, which contains the complete TAT coding
sequence(5) , was subcloned 3` of the murine sarcoma virus LTR
promoter and 5` of the SV40 polyadenylation signal in the expression
vector pMEXneo(55) . The pMEXneo plasmid (pMN) includes the
neomycin phosphotransferase gene under control of the early SV40
promoter and served as a control for these experiments. To generate
specific retroviral constructs (Fig. 1), the prokaryotic lacZ gene encoding -galactosidase was cloned into pLNSX (56) following removal of the neomycin phosphotransferase gene,
thereby generating the control vector, Bg. Construction of the
synthetic human FGF-1 gene (amino acids 21-154) has been
described and demonstrated to express active recombinant protein in
prokaryotic systems(57) . To construct the chimeric FGF-1
plasmid (Fig. 1), (hst/KS)FGF-1, two pairs of complementary,
overlapping synthetic oligonucleotides were synthesized to generate an
89-bp linker that included sequences encoding the FGF-4 signal
peptide(58) . The linker gatctttccgcagcagccgccaccATGTCGGGGCCGGGGACGGCGGCGTAGCGCTGCTCCCGGCCGTCCTGCTGGCCTTGCTGGCCCC
contained squences for both an untranslated leader (lower case) and an
open reading frame (upper case) coding for the hydrophobic signal
peptide MSGPGTAAVALLPAVLLALLAP, which was fused in-frame to the
NH
termini (MANYKK . . . ) of FGF-1
.
The untranslated leader sequence (lower case) of the linker included
both potential ribosome binding (bold lettering) and Kozak (underlined)
sequences for maximal eukaryotic translational
efficiency(59, 60, 61) . Inclusion of these
structural features, coupled with the truncated form of the growth
factor, both prevented potential translation problems encountered
previously in Escherichia coli(57) and permitted
discrimination between the endogenous murine and the transduced human
gene products. Accuracy of cloning was confirmed by restriction
endonuclease mapping and/or DNA sequence analysis.
-galactosidase (Bg). The
experimental vector (hst/KS)FGF-1 includes the human
FGF1
cDNA sequence subcloned under control of
the early SV40 promoter. The untranslated leader sequence contains a
ribosome binding site (hatched box) and Kozak consensus
sequence (open box) to maximize expression of the growth
factor. To promote secretion of FGF-1, the 22-amino acid signal peptide
sequence (KS) from FGF-4 was cloned in-frame between the Kozak sequence
and NH
termini of FGF-1. Arrows indicate
approximate location of sense and antisense primers used for PCR
amplifications of DNA and RNA preparations: N5 and N3, amplimer pair
for the neomycin phosphotransferase gene; T5 and T3, amplimer pair for
TAT; B5 and B3, amplimer pair for Bg; F5 and F3, amplimer pair for
(hst/KS)FGF-1.
Cell Culture, Transfections, and
Transductions
Primary murine fibroblasts were isolated from
day 12 C57BL/6 embryos (62) and established in culture using
DMEM (Life Technologies, Inc.) supplemented with 10% (v/v)
heat-inactivated FBS (Hyclone Laboratories) and 100 units/100 µg
penicillin/streptomycin (Life Technologies, Inc.). Population doublings
were monitored; only early, stabilized passages were used for these
studies. Routine passage of isolated cells was performed at confluence
following 0.05% trypsin, 0.5 M EDTA treatment and replating (1
10
cells/cm
). Primary cells (1
10
/cm
) were transfected with 10 µg of
either pMN or pTAT using calcium phosphate-mediated precipitation (63) followed by selection (21 days) in 800 µg/ml active
Geneticin (Life Technologies, Inc.). Transient transfection assays were
performed in which 10 µg of an HIV-LTR-luciferase reporter plasmid (6) was introduced into stable, expanded populations of either
pMN or pTAT cells (1
10
) using calcium
phosphate-mediated precipitation. Cells were lysed 24 h
post-transfection and reporter gene activity measured (Luciferase Assay
System, Promega) using a Monolight 2010 luminometer (Analytical
Luminescence Laboratory). The helper-free packaging cell line
GP+envAm12 (64) was cotransfected with either Bg or
(hst/KS)FGF-1 together with pCV108, a vector containing the early SV40
promoter directing neomycin phosphotransferase gene expression (10:1
molar ratio). Cells producing a high titer
(10
-10
colony-forming units/ml) of
amphotrophic virus were isolated by
-galactosidase expression
using FACS (65) followed by selection (2-3 weeks) in 800
µg/ml active Geneticin. Viral titration was performed by
determining the number of
-galactosidase-expressing colonies
produced by transductions of 3T3 cells (56) with serial
dilutions of supernatant from the producer line. Expression of
-galactosidase was monitored (66) with the chromogenic
substrate Bluo-gal (Life Technologies, Inc.). Subconfluent
(70-80%) primary murine fibroblasts were treated with retroviral
particles (multiplicity of infection = 1-10) obtained from
fresh (6 h) supernatant, conditioned by individual producer
lines(67) . Transduced cells were isolated by
FACS(65) , plated (5
10
cells/cm
), and expanded for subsequent analyses.
Growth Analysis
Stably transfected and
transduced cells (4 10
/cm
) were seeded
in fibronectin-coated (3 µg/cm
) 12-well plates
(Corning) and grown in DMEM supplemented with either 10% or 0.5% FBS.
Cells were washed (PBS) and fed every 3 days with fresh medium. Viable
cells (trypan exclusion) for each transfectant/transductant were
counted by hemacytometer at 3-day intervals, in duplicate, using five
separate measurements/well. Lactate dehydrogenase release assays were
used to evaluate cell death/damage according to the
manufacturer's specifications (Promega).
RNA Extraction and Analysis
The
simultaneous extraction of DNA and RNA was achieved as
described(67) . Total RNA (1.0 µg) was converted to cDNA in
a 50-µl reaction containing 2.5 µM oligo(dT), 0.5 mM dNTP, 1 mM DTT,
20 units of RNasin (Promega), and 400 units of Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Inc.), buffered with 75
mM KCl, 3 mM MgCl
, and 50 mM Tris-HCl, pH 8.3. Following incubation (15 min, 40 °C; 45 min,
37 °C), samples were heated (5 min, 95 °C) to inactivate
reverse transcriptase. Either total genomic DNA (0.1 µg) or the
reverse transcribed cDNA product (5 µl) was amplified (Perkin-Elmer
9600 Thermocycler) by the polymerase chain reaction (PCR) in a
50-µl reaction containing 0.75 µM sense and antisense
primers, 0.5 mM dNTP, and 2.5 units Taq polymerase
buffered with 50 mM KCl, 2 mM MgCl
, and
10 mM Tris-HCl, pH 8.3. In each case the PCR mixture (50
µl) was aliquoted equally (25 µl) into two separate tubes and
amplified for 28 and 35 cycles using the reaction conditions as
follows: 94 °C for 15 s, 56 °C for 15 s, and 72 °C for 2
min and 11 s. Synthetic DNA amplimers for the neomycin
phosphotransferase gene were: sense, CAA GAT GGA TTG CAC GCA GG;
antisense, CCC GCT CAG AAG AAC TCG TC. Synthetic DNA amplimers for TAT
were: sense, GGC CTG GAT CTG GAG AGC AAG AAG AAA TGGACG C; antisense,
CGG GCC TGT CGG GTC CCC TCG GGA CTG GGA GGC GGG. Synthetic DNA
amplimers for
-galactosidase were: sense, GCG GTG CCG GAA AGC TGG
CTG GAG TGC G; antisense, CCG CGA GGC GGT TTT CTC GGG CGC G. The sense
amplimer for (hst/KS)FGF-1 contains the sequence GAT CTT TCC GCA GCA
GCC GCC ACC, which is unique to the untranslated region of the
transduced human gene, thereby discriminating from endogenous murine
sequences. The sense amplimer specific for the full-length, endogenous
murine FGF-1
contains the sequence ATG GCT GAA
GGG GAA ATC AC (nucleotides 1-20). The antisense amplimer for
both the endogenous murine FGF-1
and
(hst/KS)FGF-1 genes contains the sequence GGC CAT AGT GAG TCC GAG GAC
CGC G. Synthetic DNA amplimers for murine
-actin were: sense, GTG
GGC CGC TCT AGG CAC CAA; antisense, CTC TTT GAT GTC ACG CAC GAT TTC.
Both PCR and reverse transcriptase- PCR products were analyzed by 2%
(w/v) agarose gel electrophoresis using amplification of specific
expression vectors (Fig. 1) as positive controls. Accuracy of
amplification was confirmed further by restriction endonuclease mapping
of the PCR products. Nontransfected/transduced cellular DNA/RNA served
as negative controls. In addition, reverse transcriptase-PCR performed
in the absence of reverse transcriptase was used to determine the
potential of contaminating genomic DNA in total RNA preparations.
Following agarose gel electrophoresis, PCR products were stained with
ethidium bromide and visualized with UV light. Photographs (Polaroid,
type 55) of gels were digitized using a Xerox 7650 scanner and
appropriate bands quantitated using NIH Image, version 1.58 (NIH,
Bethesda, MD). Comparative analysis of specific reverse
transcriptase-PCR products obtained from separate cell populations was
achieved following standardization to levels of amplified
-actin
mRNA from identical samples evaluated within the same gel.
Protein Extraction
Cells (1
10
/cm
) were seeded and allowed to attach (16 h)
under normal growth conditions. Cultures were washed (PBS) and fed
DMEM, supplemented with 10% (v/v) FBS (72 h). To obtain extracellular
proteins, conditioned medium was harvested, centrifuged (2,000
g, 5 min), and filtered (0.45 µm) to remove cellular
debris. Total cellular proteins were extracted from individual cell
populations as described(36) . When indicated, ammonium sulfate
(Life Technologies, Inc.) was added to 90% saturation and incubated (16
h, 4 °C) with mixing. Precipitated proteins were collected by
centrifugation (9,000
g, 30 min) and resuspended in
either 20 mM Tris-HCl, pH 7.4, 1 mM EDTA (TE) or 6.0 M guanidinium hydrochloride (GuHCl), 0.5 M DTT and
filtered (0.45 µm). To prepare nuclear extracts, individual cell
populations were trypsin collected, washed (PBS), and resuspended in
hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl
, 0.2 mM phenylmethylsulfonyl fluoride,
and 0.5 M DTT). Following swelling (10 min, 4 °C), cells
were homogenized and nuclei pelleted (3,300
g); washed
(hypotonic buffer); resuspended in 0.02 M KCl buffered with 20
mM HEPES, pH 7.9, 25% (v/v) glycerol, 1.5 mM MgCl
, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM DTT (NXB); and
identified by microscopic examination. An equal volume of 1.2 M KCl buffered with NXB was added (dropwise), and nuclei were
extracted (30 min, 4 °C) with gentle agitation in the presence of
DNase (100 units/ml), 100 µM sodium orthovanadate, and 1.5
µg/ml each aprotinin and leupeptin. Following centrifugation (30
min, 25,000
g), the supernatant containing
nuclear-associated proteins was collected.
SDS-PAGE and Limited SDS-PAGE
Analysis
Affinity-extracted proteins obtained from total
cellular extracts, isolated nuclear preparations, and processed
conditioned medium were fractionated under reducing conditions using
routine 15% (w/v) SDS-PAGE(57) . In addition, total
heparin-binding proteins recovered from conditioned medium were
resuspended in 62.5 mM Tris-HCl, pH 6.8, containing 5% (v/v)
glycerol and 2% (w/v) SDS and immediately subjected to nonreducing 15%
(w/v) limited SDS-PAGE analysis under conditions in which neither the
stacking nor resolving gel contained SDS. Gels were transferred
electrophoretically to polyvinylidene difluoride membranes
(Immobilon-P, Millipore) and incubated with an affinity-purified
polyclonal antibody (1 µg/ml) against FGF-1 (68) or a
1:1,000 dilution of a polyclonal antibody (AL72) against
TAT(69) . As a control for FGF-1 and TAT specificity, the
individual polyclonal antibodies were preincubated (16 h, 4 °C)
with a 100-fold molar excess of the corresponding recombinant
protein(57, 69) , a process that completely blocked
immunoblot staining. Membranes were probed with horseradish
peroxidase-conjugated goat anti-rabbit serum (Kirkegaard & Perry
Laboratories). Films were exposed to luminescent membranes and
developed for varying amounts of time according to manufacturer's
specifications (X-Omat, Kodak). Films were digitized and appropriate
bands semiquantitated as described for reverse transcriptase-PCR
analysis. Recombinant FGF-1 or TAT served as quantitative
immunoreactive markers for individual gel analysis.
Receptor-mediated Tyrosine
Phosphorylation
To evaluate endogenous levels of tyrosine
phosphorylation, individual cell populations were seeded (1
10
/cm
) and allowed to attach (16 h) under
normal growth conditions. Cultures were washed (PBS) and fed DMEM
supplemented (36) with serum-free hormonally defined medium.
After 48 h, cells were washed and extracted as described below. To
analyze paracrine stimulation, control (either pMN or Bg) cells (1
10
) were maintained (48 h) in serum-free hormonally
defined medium. Quiescent cells were washed (PBS) and fed (1 h, 37
°C) 48-h conditioned medium, which was obtained from individual
cell populations maintained in serum-free hormonally defined medium.
Where indicated, conditioned medium included pretreatment with 1 mM DTT. Serum-free, hormonally defined media either with or without
recombinant FGF-1 (50 ng/ml) and heparin (20 units/ml) served as
positive and negative controls (respectively) for these studies. Cells
were washed (PBS containing 100 µM sodium orthovanadate)
and lysed with 10 mM HEPES, pH 7.4, containing 1% (v/v) Triton
X-100, 0.1 M NaF, 10 mM
-glycerophosphate, 10
mM sodium pyrophosphate, 1 mM EDTA, 1 mM sodium orthovanadate, aprotinin (25 µg/ml), and
phenylmethylsulfonyl fluoride (40 µg/ml). The detergent-insoluble
extract was removed by centrifugation and the supernatant incubated
with monoclonal antiphosphotyrosine antibody (3 µg/ml, ICN) for 3 h
(4 °C) followed by precipitation (2 h, 4 °C) with a suspension
of protein A-coupled Sepharose (Pharmacia Biotech Inc.). The Sepharose
beads were washed (lysis buffer), resuspended in Laemmli buffer, and
boiled (5 min). Recovered phosphoproteins were fractionated by SDS-PAGE
and Western analyzed using polyclonal antibodies against either
phosphotyrosine (1 µg/ml), cortactin (1:400), or c-src (1:100, Santa Cruz) and chemiluminescent substrates as described
above using horseradish peroxidase anti-rabbit serum.
In Situ Immunohistochemical Analyses
To
measure the proliferation index of individual transfectants and
transductants, cells (1 10
/cm
) were
seeded and allowed to attach (16 h) under normal growth conditions.
Cultures were washed (PBS) and fed DMEM supplemented with 0.5% (v/v)
FBS for 72 h, and labeled with bromodeoxyuridine (BrdU, Sigma, 10
µM) for the final 4 h. Where indicated, culture medium
contained 30 µg/ml of an anti-FGF-1 polyclonal antibody (Sigma)
added at 0, 24, and 48 h time points. To validate the mitogenic
potential of the affinity-extracted FGF-1 complexes and transgenes, NIH
3T3 cells (1
10
/cm
) were seeded and
allowed to attach (16 h) under normal growth conditions. Cultures were
washed (PBS) and maintained (36 h) in 0.5% (v/v) FBS. Either
recombinant FGF-1 or FGF-1, heparin-extracted from intra- and
extracellular compartments, was added (24 h) in the presence of heparin
(5 USP units/ml) to quiescent cells and labeled (4 h) with BrdU.
Activation of the high molecular weight FGF-1 complexes, extracted from
the extracellular compartment, was achieved by pretreatment with 1
mM DTT. Where indicated, recombinant FGF-1 and
affinity-extracted FGF-1 preparations were preincubated (16 h, 4
°C) with 30 µg/ml polyclonal anti-FGF-1 antibody (Sigma) prior
to addition. Harvested cells were resuspended (10
cells/ml), and 0.1-ml aliquots were centrifuged (Cytospin,
Shandon-Lipshaw) onto glass microscope slides and immediately fixed (15
min) in 4% (v/v) phosphate-buffered formalin. Endogenous peroxidase
activity was exhausted by incubation (15 min) with 1% (v/v)
H
0
in methanol. Slides were incubated (37
°C) sequentially for 45 min each with three separate antibody
preparations: (a) monoclonal mouse anti-BrdU (1:100, Dako); (b) affinity-purified polyclonal goat anti-mouse
immunoglobulin (1:50, Dako); and (c) soluble complexes of
horseradish peroxidase and mouse monoclonal anti-horseradish peroxidase
(1:50, Dako). All antibody preparations were diluted in 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl and 0.05% (v/v)
Tween 20 (TTBS). Peroxidase-stained cells were developed (2-9
min) with 0.5 mg/ml 3,3`-diaminobenzidine (Sigma) and counterstained (2
min) with Mayer's hematoxylin (Sigma). Following dehydration and
mounting, slides were evaluated by light microscopy to determine the
number of nuclei staining positive (brown, 3,3`-diaminobenzidine) and
negative (blue, Mayer's). The proliferation index was determined
by dividing 3,3`-diaminobenzidine-stained nuclei by the total observed
nuclei. Two slides per condition were analyzed for individual
transductants by blinded evaluation, and at least five fields
(approximately 250 cells) per slide were counted. Statistical
significance in proliferation index were assessed using Student's t test (Abacus Concepts Inc., Berkeley, CA).
-galactosidase (FACS), were approximately the
same (30-35%) for both Bg- and (hst/ KS)FGF-1-transduced cells.
No attempt was made to subclone specific cell populations relative to
expression levels of either the selectable markers (neomycin
phosphotransferase,
-galactosidase) or modulatory transgenes (TAT,
FGF-1). A characteristic reverse transcriptase-PCR product (790 bp) was
identified (Fig. 2A) for the neomycin
phosphotransferase gene in both pMN- and pTAT-transfected cells, which
is consistent with the ability of these cells to maintain growth in
Geneticin. Expression of TAT mRNA was demonstrated by the appearance (Fig. 2A) of a predicted 278-bp reverse
transcriptase-PCR product, an observation restricted to those cells
stably transfected with the pTAT expression vector. Translation of TAT
mRNA was investigated by Western analysis (Fig. 3A),
wherein cellular (lane 2) and nuclear extracts (lane
3), as well as conditioned media (lane 4) from individual
cell transfectants were examined. Cells transfected with pTAT readily
demonstrated intracellular levels (approximately 100 ng/10
cells) of immunoreactive TAT with an apparent molecular mass of
14 kDa. The majority of TAT protein was associated with the nuclear
compartment, whereas approximately 5% of the total transgene product
was identified in the conditioned medium. Control cells transfected
with pMN failed to demonstrate the appearance of immunoreactive TAT
protein. To determine the biological activity of TAT, an
HIV-LTR-luciferase reporter plasmid was transiently transfected into
pMN and pTAT cells. More than a 100-fold increase in luciferase
activity was observed (data not shown) which was limited to cells
expressing TAT. This confirms the ability of transfected TAT to
function as a trans-activator of the HIV promoter and suggests
that primary murine fibroblasts produce the necessary cofactors to
coordinate this response.
(57) .
10
cells; lane 2) and
nuclear (1
10
cells; lane 3) extracts as
well as 48-h conditioned medium (1.2
10
cells; lane 4) of cells transfected with either pMN (top) or
pTAT (bottom) were fractionated (15%, w/v, reducing SDS-PAGE)
and analyzed using a polyclonal antibody against TAT (AL72).
Recombinant preparations of TAT (0.2 µg; lanes 1 and 5) served as positive controls. Panel B,
affinity-extracted proteins (5
10
cells) harvested
from both total cellular (lane 2) and nuclear (lane
3) extracts as well as 48-h conditioned medium (lane 4)
of cells transduced with either Bg (top) or (hst/KS)FGF-1 (bottom) were fractionated (15%, w/v, reducing SDS-PAGE) and
analyzed using the affinity-purified polyclonal antibody against FGF-1.
Recombinant preparations of FGF1
(0.2 µg; lane 1) and FGF-1
(0.2 µg; lane
5) served as positive controls. Panel C,
affinity-extracted (0.5 M NaCl) proteins harvested from both
total cellular (lane 2) and nuclear (lane 3) extracts
of cells (1
10
) transfected with either pMN (top) or pTAT (bottom) were fractionated (15%, w/v,
reducing SDS-PAGE) and analyzed using the affinity-purified polyclonal
antibody against FGF-1. Conditioned media (48 h; 5
10
cells) were processed either without (lane 4) or with (lane 5) ammonium sulfate prior to heparin-Sepharose binding.
Recombinant FGF-1
(0.1 µg; lanes 1 and 6) served as a positive control. Panel D,
affinity-extracted (0.0 M NaCl) proteins recovered from medium
conditioned (48 h) by pMN- (lanes 2 and 3) or pTAT- (lanes 4 and 5) transfected cells (5
10
) were fractionated (15%, w/v, nonreducing limited
SDS-PAGE) and analyzed using the affinity-purified polyclonal antibody
against FGF-1. Lanes 3 and 5, proteins treated (1
mM, DTT; 90 °C, 10 min) prior to gel electrophoresis.
Recombinant FGF-1
(0.2 µg; lanes 1 and 6) served as a positive
control.
A characteristic reverse transcriptase-PCR
product (355 bp) was identified (Fig. 2B) for
-galactosidase in both Bg and (hst/KS)FGF-1 cells, which is
consistent with analytical FACS and routine enzymatic analyses, which
determined that greater than 98% of expanded cells in both transduced
populations produced readily detectable levels of active
-galactosidase. Transcription of (hst/KS)FGF-1 mRNA was
demonstrated by the appearance (Fig. 2B) of a predicted
441-bp reverse transcriptase-PCR product, which was limited to those
cells stably transduced with the (hst/KS)FGF-1 retroviral vector.
Expression of (hst/KS)FGF-1 protein was investigated by Western
analyses (Fig. 3B), wherein cellular and nuclear
extracts, as well as conditioned medium, from individual cell
transductants were examined. Minimal levels (approximately 10
ng/10
cells) of immunoreactive protein (17 kDa) were
present in total cellular extracts from both Bg- and
(hst/KS)FGF-1-transduced cells, suggesting that embryonic fibroblasts
express low but detectable levels of the full-length, endogenous murine
FGF-1. As anticipated, cells transduced with (hst/ KS)FGF-1 expressed
readily detectable steady-state levels (approximately 60-80
ng/10
cells) of immunoreactive FGF-1 as a truncated
(16-kDa) protein. Analysis (Fig. 3B, lane 4)
of medium conditioned by individual transduced cell populations
demonstrated that the presence (approximately 15-20 ng/10
cells) of the FGF-1 transgene was limited to (hst/KS)FGF-1 cells,
which direct expression of the chimeric polypeptide growth factor
containing the signal peptide sequence. The apparent molecular weight
of the FGF-1 chimera approximated that of the recombinant
FGF-1
standard, suggesting accurate proteolytic
cleavage of the secreted protein. The presence of the truncated FGF-1
transgene (approximately 30-40 ng/10
cells) in
nuclear extracts (Fig. 3B, lane 3) was
restricted to (hst/KS)FGF-1-transduced cells, which is consistent with
nuclear localization requiring an exogenous pathway(41) .
Collectively, results from reverse transcriptase-PCR and Western
analyses are consistent with the engineered retroviral sequences,
thereby confirming both delivery of the nucleic acid and the ability of
primary murine fibroblasts to coordinate a transcriptional and
translational response from these templates.
10
/cm
) were examined by phase-contrast
microscopy (magnification,
100). Populations of pMN- (panels C, E, and G) and pTAT- (panels
D, F, and H) transfected cells were maintained
(48 h) in culture under normal conditions (10%, v/v, FBS). Cytospin
preparations were analyzed in situ using specific antibodies
against TAT (panels C and D), FGF-1 (panels E and F), and FGFR-1 (panels G and H).
Localization of immunoreactive polypeptides was visualized by light
microscopy (magnification,
240).
10
/cm
) were examined by phase-contrast
microscopy (magnification,
100). Populations of Bg- (panels
C and E) and (hst/KS)FGF-1- (panels D and F) transduced cells were maintained (48 h) in culture under
normal conditions (10%, v/v, FBS). Cytospin preparations were analyzed in situ using specific antibodies against FGF-1 (panels C and D) and FGFR-1 (panels E and F).
Localization of immunoreactive polypeptides was visualized by light
microscopy (magnification,
240).
When compared with respective control cells (pMN and Bg),
those constitutively expressing either TAT or (hst/KS)FGF-1 exhibited a
growth advantage under normal (10%, v/v, FBS) culture conditions (Fig. 6, A and C). When maintained in reduced
serum (0.5%, v/v, FBS), both pMN-transfected and Bg-transduced cells
demonstrated minimal growth for 9 days in culture (Fig. 6, B and D). However, growth analysis of pTAT-transfected and
(hst/KS)FGF-1-transduced cells demonstrated a significantly increased
proliferative potential. When maintained in reduced serum, each
individual cell population released minimal levels of lactate
dehydrogenase into the extracellular compartment. Normalization of
lactate dehydrogenase units to total cell number determined that less
than 2% of the individual cell populations experienced cell
death/damage. Assessment of trypan blue exclusion correlated well with
lactate dehydrogenase release assays and confirmed the absence of
sublethal cell injury for any of the individual cell populations
throughout the time course of study.
) and pTAT (
) or transduced (panels C and D) with Bg (
) and (hst/KS)FGF-1
(
). Standard error bars for each data point are within the size
of the individual symbols.
For more detailed analytical
measurements, growth kinetics were evaluated by determining the
proliferation index (BrdU incorporation) of individual cell populations
maintained (3 days) under defined culture conditions. The percentages
of pMN, pTAT, Bg, and (hst/KS)FGF-1 cells labeled under normal (10%,
v/v, FBS) conditions were very similar (40-43%). Under growth
arrest conditions (0.5%, v/v, FBS), the labeling index of control cells
(Bg-transduced) was reduced to 2.5% (Fig. 7A). In
contrast, both pTAT and (hst/KS)FGF-1 cells retained a high
proliferation index (28.5 and 25.4%, respectively), which was
significantly (p < 0.01) enhanced when compared with
control cells. Daily additions (30 µg/ml) of polyclonal antibody
against FGF-1 under conditions of growth arrest had no effect on the
labeling index of control cells. In contrast, the polyclonal anti-FGF-1
decreased the proliferation index of both pTAT and (hst/KS)FGF-1 cells
(16.2 and 11.8%, respectively), which was significantly (p < 0.01) reduced when compared with cells not exposed to the
antibody.
10
/cm
) transfected with pTAT or transduced with
Bg or (hst/KS)FGF-1 were incubated (72 h) in 0.5% v/v FBS in the
absence (white bar) or presence (black bar) of daily
additions of 30 µg/ml polyclonal antibody against FGF-1. Cells were
labeled with BrdU and analyzed as described under ``Materials and
Methods.'' Panel B, mitogenic potential of FGF-1.
Quiescent 3T3 cells (1
10
/cm
) were
treated (24 h) with affinity-extracted FGF-1 preparations (10 ng/ml),
preincubated (16 h, 4 °C) in the absence (white bar) or
presence (black bar) of the polyclonal antibody (30 µg/ml)
against FGF-1. The labeling index of FGF-1 recovered from either the
intra- or extracellular (EC) compartment of
(hst/KS)FGF-1-transduced cells was analyzed (see ``Materials and
Methods'') following BrdU incorporation. Activation of the
extracellular, high molecular weight FGF-1 complex recovered from pTAT
cells was achieved by pretreatment with 1 mM DTT. Recombinant
FGF-1
(15 ng/ml) or medium (DMEM) alone served
as a positive or negative control,
respectively.
The ability of extracellular FGF-1 and TAT to induce
similar phenotypic alterations in vitro, coupled with the
observation that antibodies against FGF-1 decreased the proliferation
index of pTAT and (hst/KS)FGF-1 cells, suggested the potential
involvement of FGF-1 in TAT-induced growth. Initial efforts utilized
reverse transcriptase-PCR analysis to investigate expression of
endogenous murine FGF-1 mRNA in each of the individual cell
populations. A characteristic reverse transcriptase-PCR product (424
bp) was identified in both transfected and transduced cell populations (Fig. 2C), and similar levels of the amplified
endogenous, murine FGF-1 mRNA product were observed in both control
transfected (pMN) and transduced (Bg) cell populations. However, when
compared with both controls, TAT-transfected and
(hst/KS)FGF-1-transduced cells demonstrated increased (1.94- and
1.37-fold, respectively) levels of endogenous, murine FGF-1 mRNA. To
correlate further TAT-induced growth with this specific polypeptide,
cellular and nuclear extracts from individual cell transfectants were
examined for the presence of endogenous FGF-1 protein. Western analysis (Fig. 3C) of total cellular extracts (lane 2)
from both pMN- and pTAT-transfected cells demonstrated similar levels
(approximately 10 ng/10 cells) of immunoreactive FGF-1
migrating as a single band (17 kDa), which is consistent with results
obtained from both Bg- and (hst/KS)FGF-1-transduced cells (Fig. 3B, lane 2). The presence of endogenous
murine FGF-1 in nuclear extracts (approximately 5 ng/10
cells) was restricted to cells transfected with TAT (Fig. 3C, lane 3), an observation consistent
with the compartmentalization of the FGF-1 transgene in (hst/KS)FGF-1
cells (Fig. 3B, lane 3). Overall,
pTAT-transfected cells demonstrated approximately a 1.5-fold increase
in total levels (intra- and extracellular) of full-length, murine
FGF-1.
cells) of
murine FGF-1 was limited to cells transfected with pTAT (Fig. 3C, lane 5). Additional efforts
determined that the complexed form of FGF-1 bound immobilized heparin
in the absence of NaCl and following elution was resolvable by limited
SDS-PAGE (Fig. 3D). In contrast to pMN cells, Western
analysis of pTAT cells demonstrated the presence of FGF-1 migrating as
multiple high molecular mass bands (Fig. 3D, lane
4), a large percentage of which displayed an apparent molecular
mass of approximately 34 kDa. Interestingly, the appearance of native
FGF-1 (17 kDa) was not detected in untreated media conditioned by
pTAT-transfected cells. Treatment of the heparin-extracted, high
molecular mass FGF-1 complexes with the reducing agent DTT (1
mM) and heat (90 °C, 10 min) generated the appearance of
native FGF-1 migrating as a single band with a representative molecular
mass (Fig. 3D, lane 5).
isolated from E. coli (Fig. 7B). Pretreatment of each growth
factor preparation with the antibody against FGF-1 significantly (p < 0.001) decreased its mitogenic potential. Correlation of
these biological data with immunoblot analysis determined that the
human FGF-1 transgene retained greater than 90% of its mitogenic
potential. In contrast, the high molecular mass, complexed forms of
full-length, murine FGF-1, affinity extracted from medium conditioned
by pTAT-transfected cells, failed to demonstrate mitogenic behavior in
the DNA synthesis assay (Fig. 5B). However, reduction
(1 mM DTT) of the heparin-extracted, high molecular mass FGF-1
complexes activated its mitogenic potential, an observation that was
inhibited significantly (p < 0.001) by pretreatment with
the polyclonal antibody against FGF-1. Semiquantitative correlation of
these biological data with immunoblot analysis determined that the
extracellular, complexed form of murine FGF-1 retained greater than 90%
of its latent mitogenic potential.
10
cell equivalents) were immunoprecipitated with a monoclonal
antibody against phosphotyrosine and Western analyzed using specific
polyclonal antibodies against either phosphotyrosine (panels A and C) or cortactin (panels B and D) as
described under ``Materials and Methods.'' Panel A,
total endogenous cellular extracts were immunoprecipitated from pMN- (lane 1) or pTAT- (lane 5) transfected cells. Control
(pMN) transfected cells were incubated (37 °C, 60 min) with either
untreated (lane 2) or treated (lane 3; 1 mM DTT) medium conditioned by pTAT-transfected cells or 10 ng/ml
recombinant FGF-1
(lane 4). Panel
B, total endogenous cellular extracts immunoprecipitated from pMN- (lane 1) or pTAT- (lane 3) transfected cells were
analyzed for the presence of phosphorylated cortactin. Lane 2,
blank. Panel C, total endogenous cellular extracts
immunoprecipitated from Bg- (lane 1) or (hst/KS)FGF-1- (lane 2) transduced cells. Control (Bg) transduced cells were
incubated (37 °C, 60 min) with either 10 ng/ml recombinant
FGF-1
(lane 3), heparin-extracted
proteins isolated from medium conditioned by (hst/KS)FGF-1 cells (lane 4), untreated conditioned medium from (hst/KS)FGF-1
cells (lane 5), or untreated conditioned medium from
Bg-transduced cells (lane 6). Panel D, total
endogenous cellular extracts immunoprecipitated from Bg- (lane
1) and or (hst/KS)FGF-1- (lane 2) transduced cells were
analyzed for the presence of phosphorylated cortactin. Approximate
sizes of induced phosphotyrosyl polypeptides were estimated using
prestained molecular mass standards (right).
Consequently,
stimulation of cell growth by FGF-1 appears to be restricted to an
extracellular pathway rather than through intrinsic interactions within
intracellular compartments.
as a biologically inactive homodimer. Indeed, the major species
detected in the novel immunoblot assay corresponds well with the
reported molecular mass representative of the FGF-1 homodimer induced
by copper oxidation(79) . As a homodimer structure, FGF-1 is
biologically inactive and requires reduction of oxidized cysteine
residues to restore complete heparin affinity, receptor binding, and
full mitogenic potential(79) . Interestingly, the HIV-1 TAT
protein has been demonstrated to form metal-linked homodimers involving
highly conserved cysteine residues(69, 80) . Since
transfected cells expressing TAT demonstrated the presence of the
transgene in both the nuclear and extracellular compartment, it
appeared possible that FGF-1 and TAT might participate in heterodimer
formation. However, Western analysis of heparin-extracted FGF-1
complexes, including those treated with DTT, failed to identify the
presence of TAT as a coextracted protein. In addition, in vitro efforts, using recombinant preparations of these two proteins
under optimal conditions for homodimer
formation(28, 69, 80) , failed to demonstrate
formation of FGF-1
TAT heterodimeric structures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.