(Received for publication, May 15, 1995; and in revised form, January 2, 1996)
From the
We reported previously that the potency of heparin-binding
fibroblast growth factor-1 (FGF-1) as a mitogen for rat hepatocytes in
primary culture is as high as that of epidermal growth factor (EGF) and
hepatocyte growth factor. To gain insight into the pathophysiological
significance of FGF-1 in hepatocyte growth, we analyzed the cooperative
mitogenicity of FGF-1 and EGF. Results from a nuclear labeling assay
using [H]thymidine suggest that most hepatocytes
in primary culture consist of two cell populations that differ in
response to FGF-1; one is an FGF-1-responsive cell population, and the
other is an EGF-responsive (but not FGF-1-responsive) cell population.
On the other hand, autoradiographic analysis of
I-FGF-1
binding demonstrated that high affinity FGF receptors were
homogeneously distributed on the surface of all hepatocytes.
Cross-linking
I-FGF-1 to the nonstimulated hepatocyte
surface indicated that the high affinity FGF receptors comprise two FGF
receptors that differ in molecular mass (128 and 79 kDa). Furthermore,
the 79-kDa receptor was preferentially down-regulated when the
hepatocytes were stimulated with EGF or hepatocyte growth factor. These
data suggest that the abundant expression of the 79-kDa FGF receptor on
some populations of hepatocytes is involved in their lack of response
to FGF-1. The 128- and 79-kDa FGF receptors were assigned as FGFR2
using an antibody specific to the ectodomain of FGFR2, whereas the
79-kDa receptor was not reactive to the antibody against the carboxyl
terminus of FGFR2. This 79-kDa FGF receptor was not
tyrosine-phosphorylated in response to FGF-1 stimulation, while the
128-kDa FGF receptor was recognized by anti-phosphotyrosine antibody
under the same conditions. Also, the heterodimer of 79- and 128-kDa FGF
receptors was less tyrosine-phosphorylated than the homodimer of
128-kDa FGF receptors. These data suggest that the 79-kDa FGF receptor
inhibits the function of the 128-kDa FGF receptor through their
heterodimerization. Thus, we surmise that the difference in response to
FGF-1 between the cell populations of normal rat hepatocytes was caused
by the different levels of the 79-kDa FGF receptor in each cell
population.
Various growth factors, including transforming growth
factor- (TGF-
), (
)epidermal growth factor (EGF),
and hepatocyte growth factor (HGF), have been identified as potent
hepatotrophic factors in primary-cultured
hepatocytes(1, 2, 3) . In agreement with the
consequence in vitro, TGF-
(which acts through the EGF
receptor) and HGF may contribute to the proliferation of liver
parenchymal cells during regeneration in
vivo(1, 2) . We previously reported that the
mitogenic activity of heparin-binding fibroblast growth factor-1
(FGF-1) in rat hepatocytes is comparable to that of EGF and HGF in
vitro(4, 5) . In addition, Kan et al.(6) reported that the expression of FGF-1 mRNA is
immediately induced in the remnant of rat liver after partial
hepatectomy, prior to the induction of TGF-
and HGF
mRNAs(7, 8) . These observations suggest that FGF-1
also promotes the growth of liver parenchymal cells during liver
regeneration; however, the significance of FGF-1 in the proliferation
of liver parenchymal cells has remained unknown. In this study, we
examined the contribution of two potent mitogens, FGF-1 and EGF, to
hepatocyte growth in vitro and found that the simultaneous
addition of FGF-1 and EGF had a spatiotemporal additive effect upon DNA
synthesis. These results suggest that the mitogenicity of FGF-1 in rat
hepatocytes is mediated by an FGF-1-responsive cell population, which
differs from an EGF-responsive cell population.
Furthermore, to clarify the cause of this difference in mitogenicity of FGF-1 between these hepatocyte subpopulations, we characterized the FGF receptors on rat hepatocytes in primary culture. The well characterized FGF receptor family consists of two categories as follows: (i) four types of high affinity FGF receptors (FGFR1, FGFR2, FGFR3, and FGFR4) that are 110-150-kDa transmembrane protein-tyrosine kinases with two or three immunoglobulin-like loops and an acidic domain in the extracellular region (9, 10) and (ii) low affinity FGF-binding sites (synonymous with heparan sulfate proteoglycans) that play an important role in the binding of FGF to the high affinity FGF receptors(10, 11) . Since exogenous heparin can mimic the function of the low affinity FGF-binding sites for FGF-1 (5) as well as FGF-2(11) , we concluded that the difference in mitogenicity of FGF-1 between hepatocyte subpopulations was not caused by the quantity and/or nature of the heparan sulfate chains on the cell surface when heparin was added to the culture medium. We therefore presume that the difference in mitogenicity of FGF-1 between hepatocyte subpopulations is due to signal transduction via the high affinity FGF receptor.
The high affinity FGF receptor is essential for growth promotion by FGF stimulation(9, 10, 12) . After binding of FGFs, the high affinity FGF receptors form a dimeric complex(13, 14) ; tyrosine kinase in the FGF receptor is activated; and then the signal for growth promotion is transduced through the phosphorylation of some proteins including the high affinity FGF receptor itself(15) . In this study, we detected two forms (128 and 79 kDa) of FGFR2 on rat hepatocytes. The 79-kDa FGF receptor was not activated by FGF-1 stimulation and inhibited the function of the 128-kDa FGF receptor. In addition, it was suggested that the 79-kDa FGF receptor was abundantly expressed in the cell population that lacked a responsiveness to FGF-1. Thus, the roles of the 79-kDa FGF receptor are discussed in relation to its involvement in the difference in mitogenicity of FGF-1 between rat hepatocyte subpopulations.
For autoradiography,
[H]thymidine (37 kBq/ml, 24.8 GBq/mmol) was added
to the cultures 30 h after cell seeding. After 24 h, the cultures were
washed with cold phosphate-buffered saline (PBS), fixed, and processed
as described(4, 5) .
Figure 1:
Time course of DNA synthesis of rat
hepatocytes in primary culture. Five hours after seeding, the cells
were incubated without (open circles) or with 10 ng/ml EGF (closed circles), 10 ng/ml recombinant human FGF-1
supplemented with 5 µg/ml heparin (open triangles), or
both growth factors (open squares). The cells were labeled for
3 h with [H]thymidine and then harvested. Each point represents DNA synthesis at the time of harvest with the
mean ± S.E. of triplicate cultures.
The populations of hepatocytes that were stimulated by various
concentrations of FGF-1 and/or EGF were determined by labeling the
nuclei with [H]thymidine. As shown in Table 1, EGF and FGF-1 individually induced
55% labeling of
hepatocyte nuclei at 10 ng/ml, the concentration at which each growth
factor exerted their maximal activities as described(4) . Even
when the concentration of each growth factor was increased up to 100
ng/ml, the labeling indices were not increased. However, the number of
nuclei labeled by a combination of these growth factors was enhanced to
80% at a 10 ng/ml concentration of each growth factor. The labeling
indices were not changed by increasing the concentration of FGF-1
and/or EGF up to 100 ng/ml. These data indicated that 55% of the
hepatocytes were responsive to FGF-1, although FGF-1 did not exert its
mitogenic activity on the remaining hepatocytes.
Figure 2:
Specific binding of FGF-1 to rat
hepatocytes in primary culture. Thirty hours after cell seeding, the
equilibrium binding of I-FGF-1 to hepatocytes was
analyzed as described under ``Experimental Procedures.'' The
background in the presence of a 200-fold excess of nonradiolabeled
FGF-1 was subtracted from the total binding in the absence of
nonradiolabeled FGF-1. Inset, Scatchard plot of FGF-1 binding
to rat hepatocytes.
For the high affinity binding sites for FGF-1
characterized above, we developed a method of detecting high affinity
FGF-1-binding sites on single rat hepatocytes in primary culture. We
added I-FGF-1 (100 ng/ml to saturate the binding of FGF-1
to its high affinity receptor) to cultured hepatocytes and then
extracted them with heparin to eliminate the low affinity binding.
After microautoradiography, grains on single cells were scored, and the
cell area was measured under the microscope (Fig. 3, A and B). As shown in Fig. 3C, the grain
density of total
I-FGF-1 binding was distributed as one
peak. This fits to the normal distribution, which had a mean value and
standard deviation of 10.36 ± 2.20 grains/100-µm
cell area. The grain density of the background was 0.84 ±
0.27 grains/100-µm
cell area, and none of the cells
corresponded to this background value. However, the grain density on
the cells was decreased to 1.70 ± 0.72 grains/100-µm
cell area when excess nonradiolabeled FGF-1 was added (Fig. 3D). The cell area associated with total binding
was 2330 ± 830 µm
, and that with nonspecific
binding was 1830 ± 680 µm
, indicating that the
areas were essentially similar. Thus, the grain density of total
binding was significantly greater than that of nonspecific binding (p < 0.01).
Figure 3:
Detection of high affinity binding sites
for I-FGF-1 on rat hepatocytes in primary culture.
Micrograph of an intact single hepatocyte showing silver grains (A and B) and histograms of their grain density (C and D) are represented. Thirty hours after seeding, the
binding of 100 ng/ml
I-FGF-1 to hepatocytes in the
presence of 50 µg/ml heparin proceeded without (A and C) or with (B and D) a 200-fold excess of
nonradiolabeled FGF-1. After heparin extraction, the cells were fixed,
and
I-FGF-1 bound to the high affinity FGF receptor was
visualized by microautoradiography. The silver grains on single
hepatocytes were scored (50 cells), and the cell area was also
measured. The bar (A and B) indicates a
10-µm scale. Vertical dashed lines (C and D) indicate the mean value of the grain density in the
background area.
Figure 4:
Effect of EGF or HGF on binding of FGF-1
to high affinity binding sites on rat hepatocytes in primary culture. A, competitive FGF receptor binding with EGF or HGF. Rat
hepatocytes were cultured in basal medium 5-30 h after cell
seeding. The binding of I-FGF-1 (0.7 nM) to its
high affinity binding sites was measured in the presence of
nonradiolabeled FGF-1 (closed circles), EGF (40 nM; open triangles), or HGF (6 nM;
). Data
represent the means ± S.D. of duplicates. B, Scatchard
analysis of FGF-1 binding to rat hepatocytes stimulated with EGF or
HGF. Hepatocytes were incubated without (circles) or with 10
ng/ml EGF (triangles) or 10 ng/ml HGF (
) 5-30 h
after cell seeding.
I-FGF-1 binding to hepatocytes
proceeded 5 h (open circles) and 30 h (closed
circles, triangles, and
) after cell seeding as
described under ``Experimental Procedure.'' B/F,
bound/free.
Figure 5:
Cross-linking of I-FGF-1 to
rat hepatocytes in primary culture. Hepatocytes were cultured without (lanes 1 and 2) or with 10 ng/ml HGF (lane
3) or 10 ng/ml EGF (lane 4) 5-30 h after cell
seeding.
I-FGF-1 bound to hepatocytes in the absence (lanes 1, 3, and 4) or presence (lane
2) of a 200-fold excess of nonradiolabeled FGF-1.
I-FGF-1 and cells were cross-linked, lysed, and resolved
by SDS-PAGE, and then the gel was visualized using a radioimage
analyzer. The molecular mass markers (in kilodaltons) are indicated on
the left. The color bar on the right indicates the
relationship between the color level and the relative intensity of the
radioactivity.
Figure 6:
Identification of FGF receptors on rat
hepatocytes in primary culture. A, immunoprecipitation of
FGFRFGF-1 complexes by antibodies to each carboxyl terminus of
FGFR1 (lane 1), FGFR2 (lanes 2 and 6), FGFR3 (lane 3), and FGFR4 (lane 4). The FGF receptors
cross-linked with
I-FGF-1 in the absence (lanes
1-5) or presence (lane 6) of excess nonradiolabeled
FGF-1 were immunoprecipitated by these antibodies. Normal rabbit serum
was used as a control (lane 5). The immunocomplexes were then
resolved by SDS-PAGE. The arrow indicates the 144-kDa
FGFR
FGF-1 complex. B, immunoprecipitation of FGFR
proteins by antibodies to each ectodomain of FGFR2 (lane 1)
and FGFR4 (lane 3). Normal rabbit serum was used as a control (lane 2). The [
S]methionine-labeled
proteins were immunoprecipitated by these FGFR antibodies, and the
immunocomplexes were resolved by SDS-PAGE. The arrow and arrowhead indicate the 128- and 79-kDa FGF receptors,
respectively. The molecular mass markers (in kilodaltons) are indicated
on the left.
We examined the
oligomerization and tyrosine phosphorylation of the two FGF receptors
to clarify whether they were involved in signal transduction by FGF-1
stimulation. After the binding of I-FGF-1 to cell-surface
FGF receptors on ice for 4 h, the cells were incubated at 37 °C for
5 min to potentially activate signal transduction pathways;
I-FGF-1 and FGF receptors were then cross-linked, and the
formed FGFR
FGF-1 complexes were adsorbed to wheat germ
agglutinin-Sepharose or immunoprecipitated by anti-phosphotyrosine
antibody. These complexes were analyzed by SDS-PAGE followed by
autoradiography. Fig. 7A shows that the dimeric form of the
FGFR
FGF-1 complexes migrated as a 220-320-kDa band together
with two monomeric forms (144- and 95-kDa bands). The broad band of the
dimeric complex corresponded to a combination of a homodimer of the
144-kDa FGFR
FGF-1 complex and a heterodimer of the 144- and
95-kDa FGFR
FGF-1 complexes. It is likely that the
radiointensities of the homodimer and heterodimer were comparable. The
homodimeric form of the 95-kDa FGFR
FGF-1 complex was not found.
These bands disappeared in the presence of excess nonradiolabeled FGF-1 (Fig. 7A). The 144-kDa band of the FGFR
FGF-1 complex
was immunoprecipitated by anti-phosphotyrosine antibody, whereas the
95-kDa band of the FGFR
FGF-1 complex was not detected by this
antibody (Fig. 7B). In addition, a 280-300-kDa
band corresponding to the homodimeric form of the 144-kDa FGFR
FGF-1 complex was strongly tyrosine-phosphorylated. The heterodimer
(220-280-kDa band) of the 144- and 95-kDa FGFR
FGF-1
complexes was recognized as a weak band by anti-phosphotyrosine
antibody. These bands were not detected when the incubation at 37
°C for 5 min was eliminated (data not shown).
Figure 7:
Characterization of FGF receptors on rat
hepatocytes in primary culture. A, dimerization of
FGFRFGF-1 complexes.
I-FGF-1 was cross-linked to
FGF receptors without (lane 1) or with (lane 2) a
200-fold excess of nonradiolabeled FGF-1. The lysates were partially
purified with wheat germ agglutinin-Sepharose and separated by SDS-PAGE
(5% gel). B, tyrosine phosphorylation of FGFR
FGF-1
complexes.
I-FGF-1 was cross-linked to FGF receptors
without (lane 1) or with (lane 2) excess
nonradiolabeled FGF-1. The lysates were incubated with
anti-phosphotyrosine antibody, and the resulting immunocomplexes were
resolved by SDS-PAGE (7% gel). Molecular mass markers (in kilodaltons)
are on the left. Apparent molecular masses (in kilodaltons) on the
right of A and B were determined by extrapolation of
data with standards. The arrow and arrowhead indicate
the 144- and 95-kDa FGFR
FGF-1 complexes,
respectively.
Some mitogens, such as TGF-, EGF, and HGF, contribute to
the proliferation of hepatocytes(1, 2, 3) .
Recently, we demonstrated that FGF-1 is also a strong mitogen for
hepatocytes in vitro(4, 5) . However, the
significance of FGF-1 in the cooperative effects with other hepatocyte
mitogens has not been addressed. In this study, we demonstrated that
two major hepatocyte mitogens, FGF-1 and EGF, additively stimulated DNA
synthesis in rat hepatocytes as follows. (i) Each growth factor had a
saturating level of stimulating DNA synthesis in hepatocytes. (ii) Even
when the DNA synthesis was stimulated up to the maximal level by one of
these growth factors, the stimulation of the other growth factor was
additive. (iii) This stimulation was additive throughout the first S
phase. (iv) The fractions of hepatocytes that responded to FGF-1, EGF,
and both growth factors were about 55, 55, and 80%, respectively. These
results indicate that 80% of the hepatocytes consist of FGF-1- and
EGF-responsive cells. Furthermore, these results suggest that the
mitogenicities of FGF-1 and EGF are mediated by the different cell
populations corresponding to each growth factor. This suggestion not
only supports the hypothesis that the EGF- and HGF-responsive cell
populations are contained in rat
hepatocytes(24, 25, 26, 27) , but
also adds a new FGF-1-responsive cell population that is detectable
under the conditions that we used.
Kan et al.(6) have demonstrated that EGF can neutralize or mask the
mitogenic effect of FGF-1 when the mitogenic activity of FGF-1 in
hepatocytes is much lower than that of EGF in serum-free cultured
hepatocytes. As we reported, however, the mitogenic activity of FGF-1
in hepatocytes is comparable to that of EGF and HGF under conditions
that may better reflect the milieu of hepatocytes in vivo(4, 5) . We demonstrated here that excess EGF
does not inhibit the FGF-1 activity and that EGF acts additively with
FGF-1. These data suggest the sequential proliferation of respective
cell populations after liver injury is due to the sequential expression
of FGF-1 and EGF/TGF-, respectively, rather than the earlier
speculation that expressed EGF/TGF-
inhibited liver parenchymal
cell proliferation that is promoted by FGF-1(6) .
We
investigated the cause of the difference in mitogenicity of FGF-1
between FGF-1-responsive and -unresponsive cell populations in rat
hepatocytes. Using a modified method of the FGF-1 binding assay, high
affinity binding sites on a single hepatocyte were detected, and the
difference in the density of these sites on each hepatocyte was
statistically analyzed. It was revealed that the silver grains indicate
the actual high affinity binding sites for FGF-1 based on the following
reasons. (i) The binding of FGF-1 was maintained after heparin
extraction and had a dissociation constant of 150-400
pM, which is remarkably different from that of the low
affinity binding of FGF-1 (K > 1.5
nM); and (ii) the grain density was significantly reduced to
the background level in the presence of excess nonradiolabeled FGF-1 (p < 0.01). In addition, since the Scatchard analysis
confirmed that >90% of the high affinity binding sites for FGF-1
were occupied by the radiolabeled ligands at the concentration of
I-FGF-1 used here, we concluded that the silver grains
represent most of the high affinity binding sites for FGF-1. If the
difference in response to FGF-1 between hepatocyte subpopulations was
caused by the presence or absence of the high affinity binding sites
for FGF-1, two peaks would be observed in the histogram of the grain
density. One peak would reflect the absence of the high affinity
binding sites for FGF-1, and the second peak would reflect their
presence. However, a peak corresponding to cells without the high
affinity binding sites for FGF-1 was not observed, and the grain
density of high affinity binding sites for FGF-1 was normally
distributed. These results suggest that the grains are homogeneously
distributed on each hepatocyte. Thus, we concluded that high affinity
binding sites for FGF-1, which are regarded as high affinity FGF
receptors, exist on the surface of all hepatocytes. It is suggested
that the difference in mitogenicity of FGF-1 between FGF-1-responsive
and -unresponsive cell populations is not due to the presence or
absence of high affinity FGF receptors.
Despite the existence of
high affinity FGF receptors in all hepatocytes, only 55% of the
hepatocytes responded to FGF-1. We then examined the effect of EGF and
HGF, which were thought to have other cell populations in
hepatocytes(24, 25, 26, 27, 28) ,
on the binding of FGF-1 to hepatocytes. We confirmed that EGF and HGF
did not interfere with the binding of FGF-1 to FGF receptors on rat
hepatocytes. After incubation with EGF or HGF for 25 h, however, the
number of FGF receptors on hepatocytes decreased to half, but the FGF-1
binding affinity was not affected. Thus, FGF receptors were
down-regulated by EGF and HGF. These results, in combination with the
implication that the mitogenicity of EGF is mediated by the
EGF-responsive cell population, which differed from the
FGF-1-responsive cell population, suggest that FGF receptors in
EGF-responsive cells are down-regulated by EGF. It is possible to
speculate that HGF induces the down-regulation of FGF receptors in
HGF-responsive cells by a similar manner to that of EGF since HGF and
FGF-1 additively stimulate the DNA synthesis of rat
hepatocytes(5) . Furthermore, the down-regulation of FGF
receptors by EGF or HGF was confirmed by cross-linking studies using I-FGF-1. A 79-kDa FGF receptor was abundantly found on
the surface of nonstimulated hepatocytes together with the 128-kDa FGF
receptor (Table 2). However, the 79-kDa FGF receptor was
preferentially down-regulated by EGF or HGF stimulation. As discussed
above, these results suggest that this down-regulation of the 79-kDa
FGF receptor occurred in EGF- and HGF-responsive cells. This raises the
possibility that, under nonstimulating conditions, the 79-kDa FGF
receptor is a dominant form in the EGF- or HGF-responsive cell
populations, these populations also being synonymous with the
FGF-1-unresponsive cell population. In contrast, the 128-kDa FGF
receptor is likely to be dominant in the FGF-1-responsive cell
population since the reduction level of the 128-kDa FGF receptor was
less than that of the 79-kDa FGF receptor when the cells were
stimulated with EGF or HGF. Therefore, it is suggested that the
abundant expression of the 79-kDa FGF receptor is involved in the lack
of responsiveness to FGF-1 in the FGF-1-unresponsive cell population.
The mRNA expression of FGFR2, but not that of FGFR1, has been
confirmed in primary-cultured rat hepatocytes(29) , and the
FGFR4 mRNA is not expressed in adult rat liver(30) . In
addition, a full-length FGFR2 (135 kDa) and an amino-terminal truncated
FGFR2 (115 kDa) with a deleted first immunoglobulin-like loop and
acidic domain have been described(9, 10) , and it has
been reported that both of these FGFR2 receptors have similar
activities in FGF-1 binding and that these FGFR2 receptors are
comparably activated by FGF-1(31) . Evidence has also been
provided that FGF-1 binds to the second immunoglobulin-like loop of
FGFR2 molecules(32) . Furthermore, many species of truncated
variants of FGFR2 in the carboxyl-terminal region have been
reported(33) . Here, we demonstrated that the 128- and 79-kDa
FGF receptors were FGFR2 using an antibody against the ectodomain of
FGFR2. The 128-kDa FGF receptor was also recognized by the antibody to
the carboxyl terminus of FGFR2; however, this antibody did not react
with the 79-kDa FGF receptor. Neither the 128- nor the 79-kDa FGF
receptor reacted with any polyclonal antibodies against the
carboxyl-terminal regions of FGFR1, FGFR3, and FGFR4. These results
indicate that the 128-kDa FGF receptor is an FGFR2 with its carboxyl
terminus intact, whereas the 79-kDa FGF receptor is a carboxyl-terminal
truncated FGFR2. In addition, the molecular mass of the 79-kDa FGF
receptor suggests that the cytoplasmic domain including the kinase
domains is likely to be deleted in this form. This is further supported
by the observation that when the cells were stimulated with FGF-1, the
79-kDa FGF receptor was not tyrosine-phosphorylated, whereas the
phosphorylation of the 128-kDa FGF receptor was detected. Thus, the
79-kDa FGF receptor is not activated by FGF-1 stimulation upon FGF-1
binding to its extracellular domain. Two types of FGFR2 (BEK and
keratinocyte growth factor (KGF) receptor) that differ in the third
immunoglobulin-like loop have been identified in human, mouse, and rat (34, 35, 36) . Although FGF-1 binds to both
types of FGFR2, KGF reacts with only the KGF receptor(37) .
Reverse transcription-polymerase chain reaction analysis of the third
immunoglobulin-like loops of FGF receptors revealed that two types (BEK
and KGF receptor) of FGFR2 mRNA were expressed in primary-cultured rat
hepatocytes, and KGF bound only to the 128-kDa FGF receptor. ()Thus, this 79-kDa FGF receptor is likely to be a BEK-type
spliced form of FGFR2, but lacking most of its cytoplasmic kinase
domain.
The tyrosine phosphorylation of the protein corresponding to the size of the homodimer of the 128-kDa FGF receptor was more intense, while that of the size of the heterodimer of the 128- and 79-kDa FGF receptors was less intense. The results suggest that the 79-kDa FGF receptor is an inactive FGF receptor, even when the heterodimers with the 128-kDa FGF receptor are formed. The data further imply that this 79-kDa FGF receptor inhibits the signal transduction from FGF receptors by the reduction of the tyrosine-phosphorylated level since it has been suggested that the signals for growth promotion by FGF-1 are transduced by the proteins that recognize the phosphotyrosine of the FGF receptor (38) . In fact, the expression of kinase-deficient FGF receptors in excess has been reported to induce the inhibition of signal transduction through the lower tyrosine-phosphorylated dimeric form of the FGF receptor(14) . Furthermore, various forms of FGFR1 (including kinase-truncated transmembrane FGFR1) have been reported to be expressed in hepatoma cells(39) , although the FGFR1 mRNA is not expressed in normal rat hepatocytes(29) . Also, Shi et al.(40) have suggested that the control of mitogenicity by different concentrations of FGF-1 in hepatoma cells is based upon spliced variants of FGFR1. Thus, we conclude that the 79-kDa FGF receptor is an inactive FGF receptor and suggest that it is abundantly expressed on the surface of an FGF-1-unresponsive cell population. In other words, the expression level of this inactive 79-kDa FGF receptor may determine the responsiveness to FGF-1 of each hepatocyte. While this speculation still remains hypothetical, it raises the possibility that the mitogenicity of FGF-1 in normal liver parenchymal cells may be controlled by a dominant-negative FGF receptor.
In summary, we have demonstrated that there are FGF-1-responsive and -unresponsive cell populations in primary-cultured rat hepatocytes and that this difference in mitogenicity of FGF-1 is likely to depend on a difference in the expression level of an inactive 79-kDa FGF receptor in individual cells. Further investigations of the expression and function of the 79-kDa FGF receptor are necessary to elucidate the pathophysiological significance of FGF-1 in growth control of liver parenchymal cells in vivo.