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Pharmaceuticals Section, Life Sciences Laboratory, Performance Materials R&D Center, Mitsui Chemicals, Inc., 1144 Togo, Mobara-shi, Chiba 297-0017, Japan
Address all correspondence and requests for reprints to: Masaru Honjo, Ph.D., Pharmaceuticals Section, Life Sciences Laboratory, Performance Materials R&D Center, Mitsui Chemicals, Inc., 1144, Togo, Mobara-shi, Chiba 297-0017, Japan. E-mail: masaru.honjo{at}mitsui-chem.co.jp
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Introduction |
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Human GH is known to bind and activate both hGH receptor (hGHR) and human PRL receptor (hPRLR) (7, 8). Previous analyses have shown that there are two hGHR-binding sites called site 1 and 2 on 22K-hGH (9). The two sites bind virtually the same site on hGHR to produce an active 1:2 (hGH:hGHR) complex (10). The complex formation proceeds sequentially, that is, the first hGHR binds to site 1 and the second one to site 2 (11). Similarly the activation of hPRLR by 22K-hGH has also been shown to proceed by a sequential dimerization mechanism (7); however, the hPRLR-binding sites on 22K-hGH do not coincide with its hGHR-binding sites (12). Particularly the three residues (His18, His21 and Glu174) in 22K-hGH are important for binding to hPRLR but not to hGHR (12, 13). In hGHR and hPRLR, an active 1:2 complex formation is followed by activation of the intracellular tyrosine kinase JAK2, which then phosphorylates signal transducer and activator of transcription (Stat) proteins such as Stat1, 3, and 5 (14, 15, 16). Tyrosine phosphorylation of Stat proteins results in their homo- or heterodimerization and translocation to the nucleus, where they induce transcription of some genes (17).
Compared with the well studied mechanism for the activation of hGHR and
hPRLR by 22K-hGH, that by 20K-hGH has not yet fully been understood,
presumably due to the difficulty for obtaining a certain amount of
20K-hGH with an authentic structure. Recently, we established an
Escherichia coli secretion system for 20K-hGH with an
authentic structure (18), and we reported that the 20K-hGH behaves as a
full hGHR agonist that forms an active 1:2 complex to the same extent
as 22K-hGH but hardly forms an inactive 1:1 complex (19, 20). With
respect to PRLR-mediated actions of 20K-hGH, several foregoing data
exist. For example, 20K- and 22K-hGH were equipotent in pigeon crop-sac
bioassay (21); in contrast, 20K-hGH had only 10.7
12.5% the potency
of 22K-hGH in Nb2 cell bioassay in the absence of Zn2+ (22, 23) and 16.5% in the binding analysis with the extracellular domain of
hPRLR (hPRLbp) in the presence of 50 µM Zn2+
(24). However, these reports have given us no information about the
effect of 20K-hGH on the full-length hPRLR.
In this paper we study 20K- and 22K-hGH in several assays such as cell proliferation, gene promoter activation, and JAK2/Stat5 tyrosine phosphorylation using transfected cells stably expressing hPRLR and human breast cancer T-47D cells.
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Materials and Methods |
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Construction of hPRLR complementary DNA (cDNA) expression
vector
On the basis of the published data (8), cDNA fragment encoding
the full-length hPRLR cDNA (nucleotides from -10 to 1877) was
amplified by RT-PCR using total RNA isolated from the human breast
cancer cell line T-47D as a template. In brief, the total RNA was
reverse transcribed with SuperScript II (Life Technologies, Inc., Gaithersburg, MD) and then amplified by KOD DNA polymerase
(Toyobo, Japan) with oligonucleotide primers hPRLR-R14
and R15, both of which have an exogenous EcoRI recognition
site (5'-CTTGAATTC-3') in the 5'-terminal. Oligonucleotide
hPRLR-R14 is 5'-CTTGAATTCGGCAGCCAACATGAAGGAAA-3', whose 20 bases in the
3'-terminal correspond to nucleotides from -10 to 10 in the hPRLR
cDNA, and hPRLR-R15 is 5'-CTTGAATTCTCAAGCTATCAGTGAAAGGA-3', whose 20
bases in the 3'-terminal are complementary to nucleotides 1858–1877.
After incubation at 94 C for 10 min, 30 cycles of reaction were
performed, each cycle consists of denaturing at 94 C for 30 sec,
annealing at 50 C for 30 sec and extension at 74 C for 1 min. The PCR
product was then digested with EcoRI and ligated to the
EcoRI-digested pCXN2 vector containing chicken ß-actin
promoter and neomycin resistant gene (25). The hPRLR cDNA sequence
ligated into pCXN2 vector was determined on DNA sequencer (ABI 373,
Perkin-Elmer Corp.) to be identical to the published
sequence and designated pCXN2-hPRLR.
Cell culture
CHO-K1 cells were cultured in DMEM containing 10% FCS, 300
µM L-proline, 4 mM
L-glutamine, 50 U/ml penicillin, and 50 µg/ml
streptomycin. Ba/F3 cells were maintained in RPMI-1640 medium
supplemented with 10% FCS, 50 µM 2-mercaptoethanol, 4
mM L-glutamine, 50 U/ml penicillin, 50 µg/ml
streptomycin, and 1 ng/ml recombinant mouse IL-3 (R&D Systems Inc.,
Minneapolis, MN). Nb2 cells were maintained in RPMI-1640 medium
supplemented with 10% FCS, 10% horse serum, 4 mM
L-glutamine, 50 U/ml penicillin and 50 µg/ml
streptomycin. T-47D cells were cultured in RPMI-1640 medium
supplemented with 10% FCS, 4 mM L-glutamine,
50 U/ml penicillin, and 50 µg/ml streptomycin, 10 µg/ml bovine
insulin (Salmond Smith Biolab Ltd., Auckland, New Zealand). Above all
cells were maintained at 37 C in 5% CO2.
Preparation of Ba/F3 and CHO cells stably expressing hPRLR
Approximately 1 x 107 Ba/F3 cells were
transfected with 50 µg of the pCXN2 vector containing hPRLR cDNA
(pCXN2-hPRLR) by being pulsed at 200 V, 960 µF in ice-cold Opti-MEM
medium (Life Technologies, Inc.). GH-responsive cells were
grown in selection medium (RPMI-1640 medium containing 10% FCS, 50
µM 2-mercaptoethanol, 4 mM
L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 1
mg/ml G418, and 10 nM 22K-hGH instead of mouse IL-3), and
resultant cells were designated Ba/F3-hPRLR. As for the preparation of
CHO cells stably expressing hPRLR, CHO-K1 cells were plated in a 100-mm
dish and transfected with 20 µg of pCXN2-hPRLR with Profection
Mammalian Transfection System (Promega Corp., Madison,
WI). Stable clones were selected in selection medium (DMEM containing
10% FCS, 300 µM L-proline, 4 mM
L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin
and 1 mg/ml G418). Among the selected clones, a clone showing the
highest binding activity to [125I]-22K-hGH was chosen and
designated CHO-hPRLR.
Confirmation of hPRLR mRNA expression by RT-PCR
Total RNAs from 1 x 107 cells of each cell
line were prepared using TRIZOL Reagent (Life Technologies, Inc.). The first-strand cDNA synthesis was performed on 3.5 µg
total RNA using reverse transcription kit Super Script II (Life Technologies, Inc.) and hPRLR-R2 primer
(5'-TCAAGCTATCAGTGAAAGGA-3'). The PCR reaction was carried out using
1/10 reverse transcription products, Vent polymerase (New England Biolabs, Inc., Beverly, MA) and two primer pairs. One primer
pair consisted of hPRLR-R12 primer (5'-TGTAAGTCACGTCCACATAA-3') and
hPRLR-R13 primer
(5'-GGCAGCCAACATGAAGGAAAATGTGGCATCTGCAACCGTTTTCACTCTGCTACTTTTTCT-3')
can amplify the 380 bp DNA fragment of 5' end of hPRLR cDNA. Another
primer pair consisted of hPRLR-R2 primer and hPRLR-R7 primer
(5'-CTAAACCCTTGGATTATGTG-3') can amplify the 367-bp DNA fragment of 3'
end of it. Aliquots of the amplified samples were separated on a 2%
agarose gel and stained with ethidium bromide and visualized by UV
light.
Preparation of 22K-hGH mutants
The authentic 22K-hGH secretion plasmid was constructed by
replacement of 20K-hGH gene segment in pGHR10 with 22K-hGH gene (18).
Three 22K-hGH multiple mutants (H18A/H21A), (K168A/E174A) and
(H18A/H21A/K168A/E174A) were obtained by a primer-introduced mutation.
The entire cDNA of three mutants were sequenced by dideoxy sequencing
methods to confirm that no additional mutations had been incorporated
during the mutagenesis. Mutants of 22K-hGH were secreted into
periplasmic fraction of Escherichia coli and purified as
described previously by Uchida et al. (18). Their sizes and
purities were evaluated by 15–25% SDS-PAGE under reducing
condition.
Cell proliferation assay
Ba/F3-hPRLR cells were cultured in the selection medium to
logarithmic phase (approximately 2 x 106 cells/ml)
and were serum starved before assay. Cells were incubated in the assay
medium (RPMI-1640 supplemented with 1% horse serum, 4 mM
L-glutamine, 50 µM 2-mercaptoethanol and
antibiotics) for 4 h and were resuspended in the fresh assay
medium at densities of 8 x 105 cells/ml. The sample
solution (50 µl) and cell suspension (50 µl) were mixed together
into the well of a 96-well plate and incubated for 18 h. When
examining zinc-mediated effect on the cell proliferation, ZnSO4 was
added to the sample solution. The measurement of cell proliferation was
achieved using a MTT assay kit (Promega Corp.) according
to the manufacturer’s protocol. In the case of proliferation of Nb2
cells, the same method was adopted with the exception of 24-h cell
starvation in the starvation medium (RPMI-1640 supplemented with 3%
horse serum, 4 mM L-glutamine, and
antibiotics).
Spi2.1 gene promoter activation assay
The construction of the luciferase reporter plasmid containing
the rat Spi2.1 gene promoter (pGL2-rSpi2.1) and the procedure for the
Spi2.1 gene promoter activation assay were described elsewhere (19).
Briefly, CHO-hPRLR were transiently cotransfected with luciferase
reporter plasmid pGL2-rSpi2.1 and ß-galactosidase reporter plasmid
pCH110 (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden)
and were incubated in serum-free DMEM medium containing various
concentrations of 20K- or 22K-hGH. After 48 h, the enzyme
activities of luciferase and ß-galactosidase were assayed.
Solubilization of cell proteins and immunoprecipitation
CHO-hPRLR cells were grown to confluence in 100-mm dishes, and
the medium was changed to starvation medium (serum-free supplemented
DMEM) 16–24 h before stimulation for 10 min by hGH. After stimulation,
cells were frozen with liquid nitrogen and lysed in 1.9 ml of cell
lysis buffer containing 10 mM Tris-HCl (pH 7.6), 5
mM EDTA, 50 mM NaCl, 30 mM sodium
pyrophosphate, 50 mM NaF, 1 mM sodium
orthovanadate, 1% Triton X-100, 1 mM PMSF, 5 µg/ml
aprotinin, 1 µg/ml pepstatin A and 2 µg/ml leupeptin at 4 C. Cell
lysate were rotated end over end for 1 h and insoluble material
was pelleted at 15,000 x g for 15 min. Supernatants
were rotated end over end for 2 h with either 2 µl of polyclonal
rabbit anti-JAK2 antibody (Upstate Biotechnology, Inc.) or
10 µl of polyclonal rabbit anti-STAT5 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then rotated for 1
h with Protein A/G PLUS Agarose (Santa Cruz Biotechnology, Inc.). The suspension was then centrifuged at 2,000 x
g, and the pellet was washed three times by cell lysis
buffer. Precipitated material was eluted off by boiling in SDS sample
buffer for 5 min, subjected to 4–12% SDS-PAGE under reducing
condition and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). In the case of T-47D
cells, the same methods were used with the following exceptions they
were cultured in 60-mm dishes and serum starved in RPMI-1640 with 4
mM L-glutamine, 50 U/ml penicillin and 50
µg/ml streptomycin.
Immunoblot analysis
Blocking of nitrocellulose membrane was done by incubating in
TBS buffer (20 mM Tris-HCl (pH 7.6), 137 mM
NaCl, 0.1% Tween 20) with 3% BSA for 1 h on rocking platform.
All steps were carried out at room temperature. The blots were then
incubated with the indicated primary antibodies in TBS buffer, washed,
and incubated with a second horseradish peroxidase conjugated goat
antimouse or antirabbit antibody (Amersham Pharmacia Biotech) in TBS buffer depending on the used primary antibody.
Immune complexes were detected by ECL chemiluminescence according to
the manufacturer’s instructions (Amersham Pharmacia Biotech). When required, membranes were stripped for 30 min at
55 C in the antibody stripping buffer (62.5 mM Tris-HCl (pH
6.7), 2% SDS, and 100 mM 2-mercaptoethanol) and reprobed
using the appropriate antibody. Quantitation of band intensity of
autoradiographs was performed using the image scanner and the analysis
software Quantity One version 2.7 (Toyobo, Japan).
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Results |
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, the single band of expected size (380
bp in the 5'-terminal fragment and 367 bp in the 3'-terminal fragment)
was detected in each lane of Ba/F3-hPRLR, CHO-hPRLR and also human
breast cancer cell line T-47D as a positive control. In contrast, no
band was detected in parental Ba/F3 nor CHO-K1 cells.
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, Ba/F3-hPRLR
cells showed a dose-dependent response to 22K-hGH; thus, the
Ba/F3-hPRLR proliferation assay was considered to be useful and
convenient for measuring hPRLR-mediated activity. To further validate
it, three 22K-hGH mutants (H18A/H21A, K168A/E174A, and
H18A/H21A/K168A/E174A), which were expected to possess decreased
binding affinity at site 1 for hPRLR based on previous reports (7, 24),
were produced and examined in this assay (Fig. 2A). As was expected,
H18A/H21A and K168A/E174A were severely damaged, and
H18A/H21A/K168A/E174A was completely damaged. These data demonstrate
that this is a useful cell line to study PRLR interactions. The
wild-type 22K-hGH and its three mutants were also examined in the rat
Nb2 cell proliferation assay, which is a common assay for measuring rat
PRLR-mediated activity. As a result, not only K168A/E174A, which was
previously demonstrated to be almost a full agonist in the Nb2 cell
assay (7), but also H18A/H21A and H18A/H21A/K168A/E174A were found to
be fully active like the wild-type (Fig. 2B). The discrepancy between
Fig. 2, A and B, although a similar one has also been raised by other
groups (7, 26), prompted us to investigate the 20K-hGH in the
Ba/F3-hPRLR cell assay in spite of already published data of 20K-hGH
examined in Nb2 cell assay (22, 23).
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shows the dose-response
curves of 20K- and 22K-hGH in the absence of exogenous
Zn2+. Both curves were bell-shaped, accounting for their
formation of 1:2 complex with hPRLR at low hGH concentrations and that
of 1:1 complex at high enough hGH concentrations. Nevertheless, the
20K-hGH curve was approximately 10-fold shifted rightward in view of
EC50 values (15 nM in 20K-hGH; 1.5
nM in 22K-hGH). According to the foregoing clinical test
(27), the concentration of serum GH after administration of 8 mg (16
IU) recombinant 22K-hGH to human subjects was less than 30 ng/ml (1.4
nM). Therefore, in combination with the result of Fig. 3A, we can raise the possibility that 20K-hGH behaves as a weaker agonist
for hPRLR than 22K-hGH in a human body when it is clinically
administered. Cunningham et al. reported that the binding
affinity of 22K-hGH for the extracellular domain of hPRLR (hPRLbp) was
increased by addition of Zn2+ (13); then we next
investigated the effect of Zn2+ on the activity of 20K-hGH
in comparison with 22K-hGH. In Fig. 3B, we fixed the GH concentration
at 2 nM because it is a maximum serum GH concentration in
the case of clinical administration to human subjects. An addition of
Zn2+ increased the activities of 2 nM 20K- and
22K-hGH; however, the enhancement was greater in 20K-hGH than in
22K-hGH. The two hGH isoforms had equal potency at 25 µM
Zn2+. It should be noted that 20K- and 22K-hGH had the same
activities at 25–100 µM Zn2+, although their
activities were rather weakened by addition of more than 25
µM of Zn2+. In human serum, free
Zn2+ concentration is reported to be less than 1
µM because the most of total Zn2+ (5–20
µM in adult population) is complexed with serum protein
such as albumin (13, 28, 29). Thus, regardless of the presence of
Zn2+ in human body, 20K-hGH may still behave as a weaker
agonist for hPRLR than 22K-hGH when administered to human.
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shows the dose-response curves of 20K- and 22K-hGH in the
presence of exogenous 25 µM Zn2+. Indeed,
20K-hGH had the same activity as 22K-hGH at low hGH concentrations
ranging from 0 to 100 nM in view of EC50 values
(0.7 nM in 20K-hGH; 0.8 nM in 22K-hGH), whereas
20K-hGH had a significantly higher activity than 22K-hGH above 100
nM.
Spi2.1 gene promoter activation caused by 20K- and 22K-hGH in
CHO-hPRLR in the absence of Zn2+
To obtain further evidence of the modest activity of 20K-hGH for
activating hPRLR, we analyzed the activities of 20K- and 22K-hGH in the
absence of Zn2+ for activating the rat serine protease
inhibitor 2.1 (Spi2.1) gene promoter because Stuff et al.
reported that rat Spi2.1 gene promoter was activated by rat
PRLR-mediated signal in rat liver (30). Hence, we constructed the
reporter plasmid containing the promoter region of rat Spi2.1 gene
linked to the coding sequence of the luciferase gene (pGL2-rSpi2.1),
and the plasmid was transiently transfected into CHO-hPRLR together
with ß-galactosidase reporter plasmid (pCH110). The luciferase
activity after 48 h incubation in the presence of 20K- or 22K-hGH
was assayed and normalized to the ß-galactosidase activity in each
transfected sample. As seen in Fig. 4, the dose-response curve of 20K-hGH was significantly shifted to a
higher concentration compared with that of 22K-hGH, which is in good
accordance with the result in Fig. 3A. As there was no increase of
luciferase activity in parental CHO-K1 cells (data not shown), the
luciferase activity induced by both hGHs were considered to be mediated
via the stably expressed hPRLR.
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, JAK2 protein signal could be detected
in each lane, although it was very weak presumably due to the weak
reactivity of anti-JAK2 antibody used in this study. The same membranes
were stripped and reprobed with antiphosphotyrosine antibody. As a
result, both 20K- and 22K-hGH caused JAK2 phosphorylation in a
dose-dependent manner; however, their patterns were quite different
(Fig. 5B). To quantitate them more precisely, the intensity of each
phosphorylation band was measured on an image scanner and an analysis
program (Fig. 5C). Consequently, the dose-response curve of 20K-hGH in
JAK2 tyrosine phosphorylation was bell-shaped and shifted to a higher
concentration compared with that of 22K-hGH.
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shows that an equal
amount Stat5 (mixture of Stat5a and Stat5b) existed in each lane. The
same membranes were stripped and reprobed with antiphosphotyrosine
antibody, then shown in Fig. 6B. The intense phosphorylation signals
were observed in both hGH isoforms; however, their patterns were
different. The intensity of each Stat5 phosphorylation signal was
quantified on an image scanner and an analysis program (Fig. 6C). The
result demonstrates that 20K-hGH causes the Stat5 tyrosine
phosphorylation at higher concentration than 22K-hGH. Tyrosine
phosphorylations of Stat1 and 3 were also tested in this system, but no
signal was observed (data not shown).
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but whose
hGHR-mediated activity was fully active (data not shown), was also
examined. Fig. 7A shows that an equal
amount of Stat5 protein existed in each lane. The same membranes
reprobed with antiphosphotyrosine antibody have revealed that there
were detectable signals in 22K-hGH (50, 150, and 500 nM)
and in 20K-hGH (500 nM). However, no apparent signal was
detected in the 22K-hGH mutant. This finding suggests that Stat5
tyrosine phosphorylation in T-47D is caused mainly via hPRLR rather
than via hGHR and that 20K-hGH is weaker than 22K-hGH in activating
endogenous hPRLR in human breast cancer cell. Tyrosine phosphorylation
of JAK2 in T-47D was also examined and similar result was obtained
(data not shown).
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12.5%
activity as compared with 22K-hGH in the absence of Zn2+ in
rat Nb2 cell growth assay (22, 23). Several groups had explained this
discrepancy by the slower metabolic clearance rate of 20K-hGH (37), the
existence of serum binding protein specific for 20K-hGH (38), or the
unique dependency of the rat PRLR-mediated activity of 20K-hGH on
Zn2+ concentration (39). However, it should be noted that
these previous data, except for that of Cunningham et al.
(24), refer to the nonhuman PRLRs-mediated activities of 20K-hGH. As
was also raised by the earlier report (26), the species specificity of
PRLR cannot be ignored in the assay of PRLR-mediated activity. Thus, we
have performed this study to clarify the effect of 20K-hGH on the
functional human PRLR and demonstrated for the first time that 20K-hGH
has only 10% hPRLR-mediated activity of 22K-hGH in view of
EC50 values of Ba/F3-hPRLR cell proliferation in the
absence of Zn2+. Furthermore, this reduced activity of
20K-hGH is enhanced to the same level as that of 22K-hGH by exogenous
25 µM Zn2+. In contrast, Cunningham et
al. reported that 20K-hGH had only 16.5% binding potency with the
extracellular domain of hPRLR (hPRLbp) in comparison with 22K-hGH in
the presence of 50 µM Zn2+ (24). The reason
for this disagreement is unclear at present; however, one possible
explanation is a putative influence of intracellular domain of PRLR on
the hormone binding affinity. According to preceding reports, 22K-hGH
binds the full-length hPRLR with Kd value of 1.8
nM in the presence of Zn2+ (7), whereas it
binds hPRLbp with higher affinity (Kd of 0.033
nM) (24). Furthermore, Ali et al. (40) have
found that the rat Nb2 PRLR (intermediate form) binds ovine PRL with
3.3-fold increased affinity than the long rat PRLR. These data strongly
suggest the involvement of the intracellular domain of PRLR in its
hormone binding. As for the reason why 20K-hGH has a lower hPRLR-mediated bioactivity than 22K-hGH in the absence of Zn2+, we speculate that the region deleted in 20K-hGH (amino acids 32–46 in 22K-hGH), may include important amino acids for interacting with hPRLR. In fact, mini-helix 1 (amino acids 42–46 in 22K-hGH) was demonstrated to be involved in the interaction of 22K-hGH with hPRLbp by crystallographic studies (12). Furthermore, Peterson et al. indicated that Phe44, which is present in all hormones stimulating PRLR-mediated action and absent in all hormones stimulating only GHR-mediated action, was critical for packing 22K-hGH in a conformation compatible with PRLR binding (41). These earlier findings consistently accounts for the modest effect of 20K-hGH on hPRLR.
In particular, it is an intriguing finding that Zn2+ enhances the potency of 20K-hGH for activating hPRLR more drastically than that of 22K-hGH. With regards to 22K-hGH, it has been already reported that 22K-hGH binds approximately 8000-fold more tightly to hPRLbp in the presence of 50 µM Zn2+ than in the presence of 1 mM EDTA (13), and that 15 µM Zn2+ enhances the potency of 22K-hGH for stimulating proliferation of FDC-P1 cells stably expressing hPRLR by about 10-fold (7), which does not agree with our result (about 2-fold). As one possibility, our culture medium might contain more endogenous Zn2+ deriving from horse serum than that of Fuh et al. Even if it is true, the endogenous Zn2+ concentration is considered to be far below 1 µM because the addition of 1 µM zinc drastically enhanced the 20K-hGH activity in the present study. According to previous reports, His18 and Glu174 in 22K-hGH are probable Zn2+ ligands (13), and His21 orients Glu174 (12). All these amino acids are retained in 20K-hGH; hence, we speculate that sufficient amount of Zn2+ might induce the conformational change of 20K-hGH site 1 region for hPRLR, which might result in the same sterical structure as that of the 22K-hGH site1 region. At any rate, further study should be done to elucidate the mechanism how Zn2+ compensates for the modest effect of 20K-hGH to the same level as that of 22K-hGH.
Close comparison of dose-response curves of 20K- and 22K-hGH in the presence of 25 µM Zn2+ in Ba/F3-hPRLR assay has revealed that 20K-hGH is higher than 22K-hGH in the cell growth activity at high hGH concentrations more than 100 nM. Similar result was also observed in our earlier study using Ba/F3 cells stably expressing hGHR, where we came to the conclusion that 20K-hGH poorly forms an inactive 1:1 complex with hGHR while it can fully form an active 1:2 complex (20). Although the stoichiometry of the complex of 20K-hGH with hPRLR still remains obscure, there is a possibility that 20K-hGH may poorly form a 1:1 complex with hPRLR in the presence of sufficient amount of Zn2+.
PRLR signal transduction is known to follow the JAK/Stat signaling pathway (14, 15, 16). PRL binding to PRLR leads to receptor dimerization, which activates PRLR-associated JAK2. Activated JAK2 phosphorylates PRLR intracellular domain, and then phosphotyrosine residues on the PRLR intracellular domain is considered to provide docking sites for the Src homology 2 (SH2) domains of Stat proteins. Tyrosine phosphorylated Stats form homo- or heterocomplexes, translocate into the nucleus, and activate PRL-inducible gene promoters including those for ß-casein (42), ß-lactoglobulin (43), interferon-regulatory factor-1 (IRF-1) (44), and Spi2.1 (30). To ensure the modest effect of 20K-hGH on hPRLR in the absence of Zn2+, the level of Spi2.1 gene activation and JAK2/Stat5 tyrosine phosphorylation in 20K- and 22K-hGH was measured and compared using CHO-hPRLR, and the resultant data are consistent with our analysis of Ba/F3-hPRLR cell proliferation.
Recently, several findings have implicated PRLR activation in the pathogenesis of breast cancer; for instance, all of the transgenic mice overexpressing rat PRL gene developed mammary carcinomas at 11–15 months of age (45), and hPRLR gene expression was increased in human breast tumors vs. normal contiguous tissues (46). These observations are highly suggestive of a role of hGH and hPRL in the development and progression of human breast cancer. Indeed, Ng et al. have reported that aged female rhesus monkeys treated with 22K-hGH for 7 weeks had 3- to 4-fold increase in mammary glandular size and epithelial proliferation index (47). In the present study, we have shown that Stat5 tyrosine phosphorylation level of 20K-hGH was evidently lower than that of 22K-hGH in human breast cancer T-47D cells. We also tested JAK2 tyrosine phosphorylation level in T-47D and obtained similar result (data not shown). Moreover, Fujikawa et al. have recently reported that 20K-hGH suppressed the T-47D tumor growth, whereas 22K-hGH promoted it in estradiol-treated nude mice (48). These evidence suggest that the mitogenic effect of 20K-hGH on human breast cancer cells could be weaker as compared with that of 22K-hGH. Take into account that 20K-hGH has the same agonistic effect for hGHR as 22K-hGH (19, 20), the modest hPRLR-mediated effect of 20K-hGH may be a great advantage in its administration to human for GH therapy because 20K-hGH may have a less life-threatening effect in mammary cancer development, whereas 20K-hGH has a full body growth effect.
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Acknowledgments |
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Received December 2, 1998.
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 M. Hayakawa, Y. Shimazaki, T. Tsushima, Y. Kato, K. Takano, K. Chihara, A. Shimatsu, and M. Irie Metabolic Effects of 20-Kilodalton Human Growth Hormone (20K-hGH) for Adults with Growth Hormone Deficiency: Results of an Exploratory Uncontrolled Multicenter Clinical Trial of 20K-hGH J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1562 - 1571. [Abstract] [Full Text] [PDF]
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 K. K. Kaulsay, T. Zhu, W. F. Bennett, K.-O. Lee, and P. E. Lobie The Effects of Autocrine Human Growth Hormone (hGH) on Human Mammary Carcinoma Cell Behavior Are Mediated via the hGH Receptor Endocrinology, February 1, 2001; 142(2): 767 - 777. [Abstract] [Full Text] [PDF]
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Y. Hashimoto, T. Kamioka, M. Hosaka, K. Mabuchi, A. Mizuchi, Y. Shimazaki, M. Tsunoo, and T. Tanaka Exogenous 20K Growth Hormone (GH) Suppresses Endogenous 22K GH Secretion in Normal Men J. Clin. Endocrinol. Metab., February 1, 2000; 85(2): 601 - 606. [Abstract] [Full Text]
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 B. Tsunekawa, M. Wada, M. Ikeda, S. Banba, H. Kamachi, E. Tanaka, and M. Honjo The Binding between the Stem Regions of Human Growth Hormone (GH) Receptor Compensates for the Weaker Site 1 Binding of 20-kDa Human GH (hGH) than That of 22-kDa hGH J. Biol. Chem., May 19, 2000; 275(21): 15652 - 15656. [Abstract] [Full Text] [PDF]
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