From the Department of Pathology, Uniformed Services
University of the Health Sciences School of Medicine, Bethesda,
Maryland 20814, § Department of Integrative Biology,
Pharmacology and Physiology, University of Texas, Houston, Texas 77030, and ¶ Department of Immunology, Lerner Research Institute, The
Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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Cytokines and hormones activate a network of
intracellular signaling pathways to regulate cell division, survival
and differentiation. In parallel, a series of growth inhibitory
mechanisms critically restrict cell population sizes. For example,
mitogens can be opposed in crowded cell cultures through
contact-inhibition or by autocrine release of antiproliferative
substances. Here, we characterize a small, heat-stable growth inhibitor
secreted by a rat T lymphoma line when cultured at high cell density.
Short term incubation (<60 min) of prolactin-responsive Nb2 lymphoma
cells at high density selectively blocked prolactin stimulation of
p42/p44 mitogen-activated protein kinases and transcription factors
Stat1 and Stat3 but not prolactin activation of Stat5 or the tyrosine
kinase Jak2. The selective effects of cell density on prolactin
signaling were reversible. Furthermore, exposure of cells at low
density to conditioned media from cells incubated at high density had
the same inhibitory effects on prolactin signaling. This selective
inhibition of discrete prolactin signals was mimicked by short term
preincubation of cells at low density with staurosporine or genistein
but not with bis-indoleyl maleimide, cyclic nucleotide analogs, calcium
ionophore A23187, or phorbol 12-myristate 13-acetate. A heat-stable,
proteinase K-resistant, low molecular weight factor with these
characteristics was recovered from high density culture medium. The
partially purified inhibitor suppressed Nb2 cell growth with a
sigmoidal concentration response consistent with a saturable,
receptor-mediated process.
Cell proliferation, differentiation, and death are controlled in a
coordinated manner by soluble factors, extracellular matrix proteins,
and direct cell-cell contact (1, 2). These extracellular regulators
influence a series of positive and negative intracellular signaling
pathways that translate into a net cellular response. Several
mechanisms for growth suppression coexist in normal cells. Direct
cell-cell contact inhibition and growth suppression through local
secretion of inhibitors work to limit proliferation (3). Although some
of these physiological mechanisms may become dysfunctional and thus
contribute to malignant transformation and tumor progression, many
remain operative in transformed cells. For example, loss of sensitivity
to transforming growth factor- Nb2 lymphoma cells are of early T cell lineage and originated in the
lymph node of an estrogenized male rat (7). These cells represent a
widely used model for PRL-induced signal transduction and gene
regulation (8-10). Despite expressing a mutant PRL receptor that lacks
an internal portion of the cytoplasmic domain (11), Nb2 cells respond
to PRL by activating multiple signaling pathways. Stimulation of the
receptor-associated tyrosine kinase Jak2 (12, 13) is followed by
activation of several signaling proteins, including latent cytoplasmic
transcription factors Stat1, Stat3, Stat5a, and Stat5b (14-17), and
the Shc-Ras-Raf mitogen-activated protein kinase (MAPK) pathway
(18-20). In addition, the Src-tyrosine kinase Fyn (21), the
phosphatidylinositol-3 kinase pathway (22), and protein kinase C (23)
have also been reported to be activated by PRL in Nb2 cells.
We now demonstrate that short term incubation of Nb2 cells at high cell
density selectively blocked PRL stimulation of p42/p44 MAPK and
transcription factors Stat1 and Stat3 but did not affect PRL activation
of Stat5 or Jak2 tyrosine kinase. This response to cell crowding was
reversible and was mimicked at low cell density by short term exposure
to conditioned high density medium, staurosporine or genistein. A low
molecular weight, heat-stable, and proteinase K-resistant factor with
these characteristics was recovered from conditioned high density
medium. Of particular importance, the partially purified factor also
inhibited cell growth in a saturable dose-dependent
fashion, suggesting operation through a receptor-mediated mechanism.
Materials--
Ovine PRL (NIDDK-oPRL-19, AFP-9221A) was supplied
by the National Hormone and Pituitary Program, NIDDK, the NICHD, and
the U.S. Department of Agriculture. Polyclonal rabbit antisera specific to peptides corresponding to the unique COOH termini of Stat1 Cell Culture and Treatment--
The Nb2 cell line (7) was
originally developed by Dr. Peter Gout (Vancouver, Canada), the Nb2-SP
clone used in this study was generously provided by Dr. Henry Friesen
(Manitoba, Canada). Cells were grown in RPMI 1640 medium (Mediatech,
catalog number 15-040-LM) containing 10% fetal calf serum (Intergen,
catalog number 1020-90), 2 mM L-glutamine, 5 mM HEPES, pH 7.3, and penicillin-streptomycin (50 IU/ml and
50 µg/ml, respectively), at 37 °C with 5% CO2. Before use in PRL stimulation experiments, Nb2 cells at a density of 1-1.5 × 106/ml were incubated for 20-24 h in
PRL/lactogen-free medium consisting of RPMI 1640, which instead of 10%
fetal calf serum contained 1% gelded horse serum (Sigma, catalog
number H-1895), hereafter referred to as starvation medium. At the
beginning of each experiment, cells were concentrated to the desired
density and incubated for the various periods as indicated in the
presence or absence of 100 nM ovine PRL during the final 15 min. Cell pellets were frozen at Solubilization of Proteins, Immunoprecipitation, and
Immunoblotting--
Frozen pellets from 1 × 108 Nb2
cells were thawed on ice and solubilized in 1 ml of lysis buffer
containing 10 mM Tris-HCl, pH 7.6, 5 mM EDTA,
50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate,
1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin. Cell
lysates were rotated end over end at 4 °C for 60 min, and insoluble
material was pelleted at 12,000 × g for 30 min at
4 °C. Depending on the experiment, clarified lysates were incubated
rotating end over end for 3 h at 4 °C with polyclonal rabbit
antisera (3 µl/ml) to individual Jak2 and Stat proteins as specified.
Antibodies were captured by incubation for 60 min with protein
A-Sepharose beads (Amersham Pharmacia Biotech, catalog number
17-0780-01), and proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and immunoblotting as described previously (17), using
polyvinylidene difluoride membranes (Millipore, catalog number 1PVH
00010) and horseradish-conjugated secondary antibodies in conjunction
with enhanced chemiluminescence substrate mixture (Amersham Pharmacia
Biotech, catalog number RPN2106) and x-ray film.
Preparation of Conditioned High Density Medium (HDM)--
Nb2
cells that had been cultured in PRL-free starvation medium for 20-24 h
at a density of 1-1.5 × 106 cells/ml were brought to
a high cell density of 4.0 × 108 cells/ml and
incubated for 1 h in the same culture medium at 37 °C. The
cells were discarded after centrifugation at 3,000 × g
for 5 min at 4 °C, and the conditioned medium was passed through a
0.22-µm filter and used fresh unless indicated otherwise. For some
experiments, conditioned HDM was boiled for 10 min, followed by
incubation on ice for 30 min with subsequent clarification by
centrifugation at 12,000 × g for 30 min at 4 °C to
remove precipitated proteins. For dialysis experiments, dialysis
membranes with a molecular weight cutoff of 10 kDa (Schleicher & Schuell, catalog number 25310) were used, and the dialysis medium was
either 100 volumes of starvation medium for removal of low molecular
weight material or four volumes of water for recovery of the low
molecular weight fraction.
Cell Proliferation Assays--
Nb2 cells were dispensed into
96-well microtiter plates at 1 × 105 cells/100 µl
and exposed for 48 h to either control medium or conditioned HDM
in some experiments or in dose-response experiments to various
dilutions of the reconstituted low molecular weight material derived
from boiled and clarified HDM. The number of viable cells were measured
metabolically by the Promega cell proliferation assay using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) as
described previously (25). Colorimetric analysis was performed using a
semiautomatic plate reader from Dynatech Laboratories (MR 600) at
absorbance 590 nm.
Selective Inhibition at High Cell Density of PRL-inducible
Activation of Stat1, Stat3, and ERK1/2 but not Jak2 and Stat5--
To
examine the effect of cell crowding on PRL-induced signal transduction,
Nb2 cells were incubated at increasing concentrations (range
0.25-4.0 × 108 cells/ml) for 45 min, with or without
prolactin (100 nM) added during the final 15 min. Cells
were maintained in suspension by manual agitation every 5 min during
the entire incubation period. PRL-inducible tyrosine phosphorylation of
Jak2, Stat1, Stat3, and Stat5 was subsequently analyzed by
immunoblotting of immunoprecipitated proteins from detergent cell
lysates. Parallel samples of whole cell lysates were also immunoblotted
with anti-phospho-MAPK antibodies to detect activated ERK1 and ERK2.
Immunoblotting with anti-phosphotyrosine antibodies or
anti-phospho-specific MAPK antibodies showed that PRL-inducible
activation of Stat1, Stat3, and ERK1/2 was significantly inhibited by
increasing cell density, particularly at 2-4 × 108
cells/ml (Fig. 1, lanes g-j,
panels 3, 5, and 7 as
numbered from the top). In contrast, PRL-inducible tyrosine
phosphorylation of Jak2 and Stat5 remained unaffected by cell density
(Fig. 1, lanes a and b versus
i and j, panels 1 and 9).
In parallel, basal levels of ERK1/2 activation were also significantly
suppressed under high cell density conditions (Fig. 1). Reblotting of
the same or duplicate samples for protein levels did not reveal any systematic changes in protein levels during the 45-min experiment period that could explain the marked reductions in signals by either
Stat1, Stat3, or MAPK.
Previously, we had established by electrophoretic mobility shift assays
that PRL-activated Stat5 and Stat1 from Nb2 cells form distinguishable
complexes with an oligonucleotide probe derived from the rat The Selective Inhibition at High Cell Density of PRL Activation of
Stat1, Stat3, and ERK1/2 Is Rapid and Reversible--
Having
demonstrated that a selective uncoupling of certain PRL-induced signals
occurred at high cell density, we examined how rapidly this inhibition
of PRL-inducible Stat1 and Stat3 tyrosine phosphorylation occurred, and
whether the inhibitory effect could be reversed by dilution of the
cells. Incubation of cells at high density (4.0 × 108
cells/ml) for varying times up to 75 min, with or without PRL added
during the final 15 min of each period, showed that whereas PRL-inducible Jak2 tyrosine phosphorylation was unaffected by cell
crowding over the entire period, PRL-inducible tyrosine phosphorylation of both Stat1 and Stat3 was significantly inhibited after 30 min and
completely after 75 min (Fig.
2A).
We also investigated whether this inhibition of discrete PRL signals
was reversible upon dilution of cells. Nb2 cells were preincubated at
high density (4.0 × 108 cells/ml) for 30 min,
followed by either continued incubation at high cell density or
dilution to low cell density (0.5 × 108 cells/ml) for
15 min in the presence or absence of PRL. Parallel control cells were
also incubated at low cell density for the entire 45-min period with or
without PRL during the final 15 min (Fig. 2B, lanes
a and b). PRL-inducible tyrosine phosphorylation of
Stat1 and Stat3 and ERK1/2 activation, but not Jak2 and Stat5 tyrosine
phosphorylation, was significantly inhibited as observed previously
when incubated at high density for 45 min (Fig. 2B, panels 1-5, lanes a-d). However, this selective
inhibition of PRL-inducible Stat1, Stat3, and ERK1/2 phosphorylation
was completely reversed by dilution for 15 min to low density after
preincubation for 30 min at high cell density (Fig. 2B,
panels 2-4, lanes e and f).
Reblotting of samples for protein expression levels were consistent
with the previous data shown in Fig. 1, demonstrating that
density-induced inhibition of Stat1, Stat3, and ERK1/2 signals could
not be explained by systematic reductions in protein levels (data not
shown). Based on these experiments, we conclude that the inhibitory
effect of cell crowding on discrete PRL signals was rapid and
reversible. Because of the rapid reversal of signal suppression upon
dilution to low cell density, and the short time at high cell density
needed for establishing the inhibitory effect, we favored a direct
mechanism that exclusively involved cell-cell contact. However, it
remained possible that the effect of cell crowding also included the
release of a soluble suppressor substance.
A Low Molecular Weight Factor in Conditioned High Density Medium
Selectively Modulates PRL Signals--
To directly test whether a
soluble mediator could account for the observed effects, we analyzed
the effect of conditioned HDM on PRL signals in cells incubated at low
density (0.5 × 108 cells/ml). HDM was generated by
incubating parallel cultures of Nb2 cells at high density (4.0 × 108 cells/ml) for 60 min at 37 °C followed by removal of
cells by centrifugation. Contrary to our expectation, short term
incubation of cells at low cell density in freshly generated HDM also
led to selective suppression of PRL-inducible tyrosine phosphorylation of Stat1 and not of PRL-inducible Jak2 activation (Fig.
3A, lanes a-f).
These observations suggested that a soluble factor was indeed present
in freshly generated HDM that could mediate the suppression caused by
cell crowding and showed that increased cell-cell contact per
se was not required to achieve the inhibitory effect. Furthermore, these experiments showed that the soluble inhibitory activity was
stable, because the activity persisted in HDM that had been stored for
24 h at 4 °C (Fig. 3A, lanes g and
h) and in HDM that had been boiled for 10 min followed by
centrifugation at 12,000 × g for 30 min (Fig.
3A, lanes i and j). On the other hand,
the soluble inhibitory activity was lost upon dialysis of boiled and clarified HDM against 100 volumes of starvation medium at 4 °C for
12 h using a molecular weight cutoff of 10 kDa (Fig.
3A, lanes k and l). Collectively,
these results indicated that a heat-stable low molecular weight factor
present in HDM could mediate the inhibition of discrete PRL signals
induced by cell crowding.
We then tested whether this small heat-stable factor could be recovered
from the dialysis medium. Conditioned HDM was boiled, clarified, and
dialyzed against 4 volumes of water overnight at 4 °C using a
molecular weight cutoff of 10 kDa. The dialysate was discarded, and the
resulting dialysis fluid containing low molecular weight material was
lyophilized and restored with water to a stock concentration
corresponding to 10 × HDM. This concentrated low molecular weight
fraction of HDM was diluted 1:10 in cell culture medium and
specifically tested for its ability to affect PRL activation of Jak2,
Stat1, and ERK1/2. In control samples, incubation at high cell density
was associated with inhibition of PRL-inducible Stat1 and ERK1/2 but
not Jak2 signals (Fig. 3B, lanes a-d). Likewise,
cells incubated at low cell density in medium containing the
reconstituted low molecular weight fraction of HDM also showed
selective inhibition of PRL-inducible phosphorylation of Stat1 and
ERK1/2 without affecting Jak2 phosphorylation (Fig. 3B,
lanes e and f). In addition, this reconstituted
dialysis medium inhibited basal activity of ERK1/2 (Fig. 3B,
panel 3, lane e), thus also mimicking this
particular effect of cell crowding. On the other hand, no inhibition
was seen in cells incubated in control medium containing the low
molecular weight fraction from low density starvation medium, which had
also been boiled, clarified, dialyzed, and reconstituted
correspondingly (Fig. 3B, lanes g and
h). We conclude that a small heat-stable activity that
specifically mimicked the effect of cell crowding could be recovered by
dialysis of HDM that had been boiled and clarified.
To address whether the inhibitory factor present in boiled and
clarified HDM was a peptide, we tested its sensitivity to proteinase K,
a general endoprotease that cleaves nonselectively after any hydrophobic, aliphatic, or aromatic amino acid (28). For these experiments, boiled and clarified HDM or corresponding control medium
(pH 7.3) were incubated with proteinase K (50 µg/ml) for 1 h at
37 °C, followed by reboiling for 10 min to inactivate the enzyme.
Consistent with the previous data, preincubation of cells with boiled
and clarified HDM suppressed the PRL-inducible Stat1 signal (Fig.
3C, lanes e and f) when compared with
pretreatment with control medium from low density cultures that had
been boiled only, or boiled and treated with proteinase K (Fig.
3C, lanes a-d). However, treatment of boiled and
clarified HDM with proteinase K did not neutralize the endogenous
inhibitor (Fig. 3C, lanes g and h). As
a positive control to verify that the proteinase K used was
enzymatically active in this biochemical setting, conditioned low
density medium was spiked with prolactin (10 nM) and
incubated for 30 min at 37 °C in the presence or absence of
proteinase K. As shown in Fig. 3D, PRL-induced tyrosine
phosphorylation of Jak2 was abolished after proteinase K treatment
(lane c versus lane d). Because proteinase K
cleaves peptide bonds after more than half of the natural amino acids
(28), these observations suggested that the low molecular weight factor
may not be a peptide. However, further physicochemical analysis and
purification is needed to determine the structure of this factor.
Modulation of Discrete PRL Signals in Nb2 Cells at High Cell
Density Is Mimicked by Protein Kinase Inhibitors Staurosporine and
Genistein--
We addressed the potential mechanism of action of the
high cell density-associated inhibitor by screening a series of
pharmacological agents for modulatory effects on PRL-inducible Jak2,
Stat1, and Stat3 signals in Nb2 cells. Cells were maintained at low
density and were preincubated with pharmacologically active
concentrations of each agent for 30 min before PRL was added for
another 15 min. The results of these studies are presented in Fig.
4, A and B, and
showed that the broad spectrum protein kinase inhibitor, staurosporine (500 nM), specifically mimicked the effect of cell crowding
by inhibiting PRL-induced tyrosine phosphorylation of Stat1 and Stat3, while having little or no effect on Jak2 activation (Fig.
4B). In contrast, the more specific protein kinase C (PKC)
inhibitor, bis-indolelyl-maleimide (500 µM), did not
affect PRL signals, suggesting that the effect of staurosporine is not
mediated by inhibition of PKC. On the other hand, genistein (200 µM), a tyrosine kinase inhibitor, also inhibited
PRL-induced Stat1 and Stat3, albeit not as markedly as staurosporine
(Fig. 4B). No effect was observed after pretreatment with
any of the other agents, which included the calcium ionophore A23187 (1 µM), 8-Br-cAMP (1 mM), 8-Br-cGMP (1 mM), the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (100 µM), and the protein kinase C
activator phorbol 12-myristate 13-acetate (1 µM).
Furthermore, the mimicry of cell crowding by staurosporine and
genistein also included resistance of PRL-inducible tyrosine
phosphorylation of Stat5a and Stat5b (Fig. 4C), as well as
sensitivity of basal and PRL-inducible ERK1/2 activation as recently
reported by us and others (29, 30). Thus, staurosporine and to some
extent genistein mimicked cell crowding modulation of PRL signals,
whereas a series of other agents had no effect. It is therefore
possible that the soluble inhibitor acts by a mechanism shared by
staurosporine and genistein to regulate PRL signals. Because
staurosporine inhibits Nb2 cell growth and survival (26, 30), we tested
whether the factor released during cell crowding also inhibited growth
of Nb2 lymphoma cells.
The Low Molecular Weight Factor in Conditioned High Density Medium
Inhibits Nb2 Cell Growth--
We first determined whether complete
conditioned HDM inhibited the growth of Nb2 cells. Nb2 cells were
cultured at a density of 106 cells/ml in control medium or
in conditioned HDM for 48 h. At that point, the number of
metabolically active and viable cells were assessed using the MTT cell
proliferation assay (25). Under these conditions, HDM significantly
inhibited cell growth by approximately 60-65% (n = 3;
p < 0.0001; Fig.
5A). Based on these
observations, we specifically tested the partially purified factor
obtained after dialysis of boiled HDM for its growth inhibitory
activity. It was of particular importance to establish whether this
partially purified activity exerted a saturable
concentration-dependent effect and was not simply
cytotoxic. For these experiments, three independently obtained stock
solutions containing the partially purified factor at a concentration
corresponding to 10× HDM were tested separately. The solutions were
diluted in culture medium to achieve concentrations corresponding to a
range of 0.25-2× HDM. Nb2 cells were then incubated for 48 h
with increasing concentrations of the reconstituted low molecular
weight material derived from either conditioned HDM or from low density
control medium, and cell growth rate was assayed by the MTT method. As
shown in Fig. 5B, the inhibitory effect of the soluble
extract displayed a sigmoidal growth inhibition curve reaching a
plateau at approximately 65%. These observations therefore strongly
suggested that the inhibitory activity released during cell crowding
operated through a saturable process.
This study describes a small heat-stable growth inhibitor secreted
by a rat T lymphoma line when cultured at high cell density. The data
also suggested that this inhibitor selectively and reversibly suppressed some, but not all, signaling pathways downstream of PRL
receptors. Specifically, short term incubation of Nb2 lymphoma cells at
high density blocked PRL stimulation of mitogen-activated protein
kinases ERK1/2 and transcription factors Stat1 and Stat3 but did not
affect PRL activation of Stat5 or the tyrosine kinase Jak2. These
effects of cell crowding were reversible and could be imitated by
incubation of cells in conditioned high density culture medium at low
cell density. A heat-stable and proteinase K-resistant low molecular
weight factor (<10 kDa) with these characteristics was recovered from
the conditioned high density medium. This factor suppressed basal and
PRL-induced MAPK activation and inhibited cell growth with a sigmoidal
concentration response consistent with a saturable mode of action.
Common Mechanism of Discrete Signal Modulation by Cell Crowding,
Staurosporine and Genistein?--
The discriminating suppression of
PRL signals by the growth inhibitor present in conditioned high density
medium could under low density culture conditions be specifically
mimicked by the broad spectrum protein kinase inhibitor, staurosporine.
Short term (30 min) pretreatment of cells with staurosporine was
associated with selective inhibition of PRL-inducible Stat1, Stat3, and
ERK1/2 signals, with little or no effect on PRL-inducible Stat5 and
Jak2 signals. A lesser but similar selective inhibition of PRL signals was induced by the more specific tyrosine kinase inhibitor, genistein. In contrast, PRL signals were not modulated by pretreatment of cells
with the PKC inhibitor bis-indoleylmaleimide, the PKC activator phorbol
12-myristate 13-acetate, the calcium ionophore A23187, or several
cyclic nucleotide mimetics. Collectively, these pharmacological data
raise the possibility that cell crowding, staurosporine, and possibly
genistein modulate PRL signals through a common and PKC-independent
mechanism. One simple model would involve inhibition by staurosporine,
genistein, and cell crowding of a tyrosine kinase that acts downstream
of Jak2 and is required for PRL-inducible activation of Stat1, Stat3,
and MAPK. Such a role may be played by one or more Src tyrosine
kinases. Both staurosporine and genistein inhibit tyrosine kinases of
the Src family (30, 31), and Fyn and possibly Lck is activated by PRL
in Nb2 cells (21). In parallel studies of Shc, an adaptor protein that
we have shown to couple PRL-receptors to the Grb2-Sos-Ras-MAPK pathway
in Nb2 cells (19), supported this notion. PRL-inducible tyrosine
phosphorylation of Shc was also inhibited after pretreatment with high
cell density, staurosporine, or
genistein.3 We are currently
testing the possibility that Fyn or other Src tyrosine kinases
represent common targets for cell crowding, genistein, and
staurosporine in Nb2 cells.
A tyrosine kinase acting downstream of Jak2 might critically mediate
PRL-induced activation of Stat1/3 and the Shc-Ras-MAPK pathway by two
alternative mechanisms. On the one hand, Stat1, Stat3, and Shc could be
direct substrates for a density-regulated tyrosine kinase.
Alternatively, this putative kinase could phosphorylate tyrosyl docking
sites within the receptor complex that are critical for bringing
Stat1/3 and/or Shc within reach of the catalytic domain of the Jak2
kinase. Future examination of the sensitivity of specific
phosphorylation sites within the prolactin receptor to inhibition by
high cell density, staurosporine, and genistein may therefore be highly informative.
Another question that we have addressed is whether density-associated
inhibition of MAPK in Nb2 cells may selectively suppress tyrosine
phosphorylation of Stat1 and Stat3 but not Stat5. We recently
demonstrated that the MEK-1 inhibitor, PD98059, blocked prolactin-induced MAPK activation in Nb2 lymphocytes without affecting inducible Stat5a and Stat5b tyrosine phosphorylation (29). However, further investigation showed that PD98059 did not inhibit
prolactin-induced tyrosine phosphorylation of either Stat1, Stat3, or
Stat5, despite effectively inhibiting MAPK activation.3
These experiments therefore did not suggest any causal link between MAPK inhibition and the selective inhibition of Stat1/3 tyrosine phosphorylation at high cell density but rather indicated that the two
inhibitory events occur in parallel.
A final mechanistic matter relates to the observed lack of sensitivity
of PRL-induced Jak2 tyrosine phosphorylation to kinase inhibitors. The
specific resistance of Jak2 tyrosine phosphorylation to both
staurosporine and genistein in Nb2 lymphocytes may suggest that Jak2
per se is not sensitive to these inhibitors. To our knowledge, the sensitivity of Jak2 to staurosporine or genistein has
not been analyzed under stringent conditions in cell-free Jak2 kinase
assays. The observation that staurosporine may inhibit cytokine-induced
Jak2 tyrosine phosphorylation in some cellular models, e.g.
growth hormone-induced Jak2 phosphorylation in fibroblasts (32), could
reflect inhibition of one or more secondary tyrosine kinases that in
certain cell lines contribute significantly to Jak2 tyrosine
phosphorylation on multiple sites.
Context-sensitive Modulation of Discrete Signaling Pathways
Downstream of PRL Receptors--
The extracelluar matrix is an
important external modulator of cell fate (1) and is known to influence
PRL signaling. Specifically, for PRL to activate Stat5 and induce
lactogenic differentiation of mammary epithelial cells, a
laminin-containing extracellular matrix was required that suppressed a
prolactin receptor-associated protein tyrosine phosphatase (33, 34).
The current observations on Nb2 lymphoma cells extends this phenomenon
by describing a discriminative pattern of external regulation of
individual signal transduction pathways downstream of PRL receptors.
Provided that the observed changes in signaling repertoire have
biological consequences for Nb2 cells, the modulated pathways may be
viewed as sensors of the degree of cell crowding. The fact that
significant quantitative changes occurred in some, but not all, PRL
signals at different cell densities may also be directly linked to the
associated growth inhibition. Although such a causal relationship
remains to be proven, the observed suppression of MAPK activities may
represent the most critical growth inhibitory component. Furthermore,
the rapid uncoupling or heterologous desensitization of discrete PRL signals by an autocrine factor during cell crowding may reflect only
one of many commonly operating mechanisms of external modulation of
particular transduction pathways. Increased awareness of
context-sensitive signaling pathways should also caution against
simplistic interpretations of signaling data.
The observed resistance of Jak2 and Stat5 to inhibition at high cell
density solidifies the Jak2-Stat5 axis as a distinct pathway as
proposed by Groner and colleagues (35, 36). We have also recently shown
that selective stimulation of Jak2 in Nb2 cells by the anti-apoptotic
drug, aurintricarboxylic acid (ATA), exclusively led to activation of
Stat5a and Stat5b, but not of Stat1 and Stat3 (26). Likewise, in this
study, staurosporine treatment affected PRL-inducible activation of
Stat1 and Stat3 but not Jak2 and Stat5. Therefore, accumulating
evidence suggests that PRL activation of Jak2-Stat5 can be
physiologically and pharmacologically dissociated from PRL activation
of Stat1 and Stat3 in Nb2 cells. However, a clearer understanding of
the biological roles of each of these pathways is needed to shed light
on their involvement in disease progression and to establish a more
specific pharmacological basis for their manipulation.
Characterization of the High Density Medium-associated
Inhibitor--
Thus far, the majority of growth inhibitory factors
isolated from cultured cells have been thermolabile polypeptides,
including transforming growth factor- Relevance of the Endogenous Growth Inhibitor to Cancer
Therapy--
Could identification of this soluble inhibitor from Nb2
lymphoma cells cultured at high density lead to new drugs against cancer? The success of antiproliferative interferons as adjuvant treatment for several types of malignancies has demonstrated the usefulness of natural growth inhibitors in cancer therapy (40). The
observations that the endogenous inhibitor suppressed MAPK activities
and may operate through a mechanism shared by staurosporine are
encouraging. MAPKs are hyperactive in a large number of tumors (41),
and at least two staurosporine analogues, 7-hydroxystaurosporine and
N-benzoylstaurosporine, are currently in clinical trials for cancer treatment (42, 43). Because staurosporine inhibits a variety of
protein kinases and induces apoptosis in many cell types (44, 45), more
selective analogues may have fewer adverse effects and be less toxic.
It will therefore also be important to establish to what extent the
endogenous inhibitory activity described here will mimic all of the
effects of staurosporine. A possible antiproliferative effect on cells
of lymphoid origin would also be of direct relevance for pharmacologic immunosuppression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, an autocrine growth inhibitor, is
frequently observed in malignant cells (4), whereas contact-inhibition
or cell density-induced growth inhibition may remain intact (5, 6).
During studies of
prolactin(PRL)1-induced
signal transduction in rat Nb2 lymphoma cells, we observed that cell
crowding selectively inhibited some but not all PRL-inducible signals,
suggesting the existence of a cell density-dependent mechanism for modulation of discrete PRL signals.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Stat3, Stat5a, and Stat5b were generated as described previously (17,
24). In addition, a polyclonal rabbit antiserum that recognized both
the 92-94-kDa
-forms and the COOH-terminally truncated 82-84-kDa
-forms of Stat5a/b was raised against a peptide derived from the
originally published sequence of sheep Stat5a as described previously
(17). Each antiserum was useful for detection of rat Stat proteins by
immunoprecipitation and immunoblotting. Monoclonal anti-phosphotyrosine
antibody 4G10 and polyclonal rabbit antiserum to Jak2 were purchased
from Upstate Biotechnology (catalog numbers 05-321 and 06-255, respectively). Affinity-purified rabbit antibodies to active MAPK were
purchased from Promega (catalog number V667A).
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Selective inhibition at high cell density of
PRL-inducible phosphorylation of Stat1, Stat3, and ERK1/2 but not Jak2
and Stat5. Nb2 cells were incubated at 37 °C for a total of 45 min at various cell densities before stimulation with (+) or without
( ) 100 nM of ovine PRL during the final 15 min. Clarified
cell lysates were immunoprecipitated with antibodies to Jak2, Stat1
,
Stat3, and Stat5 and subsequently blotted with anti-phosphotyrosine
antibodies (
-PY). Parallel samples representing whole
cell lysates were also immunoblotted with anti-phospho-MAPK antibodies
(
-pMAPK) to detect activated ERK1 and ERK2. In addition,
reblotting of the same or parallel samples for protein levels were
performed as indicated.
-casein
promoter, whereas PRL-activated Stat3 does not bind to this DNA
sequence (17, 26, 27). Parallel electrophoretic mobility shift analyses
of Stat1 and Stat5 binding to this
-casein promoter probe showed a
corresponding selective inhibition by cell crowding of PRL-inducible
Stat1 activation.2
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Fig. 2.
The selective inhibition at high cell density
of PRL activation of Stat1, Stat3, and ERK1/2 is rapid and
reversible. A, parallel samples of Nb2 cells were
incubated at low density (0.5 × 108 cells/ml) and/or
high density (4 × 108 cells/ml) for a combined period
of 75 min at 37 °C with (+) or without ( ) 100 nM ovine
PRL added during the final 15 min. At variable times before the end of
the 75-min incubation period, cells were switched from low density
(0.5 × 108 cells/ml) to high density (4 × 108 cells/ml) by centrifugation and volume reduction, so
that the individual cell samples were crowded for periods ranging from
0 to 75 min as indicated. Clarified cell lysates were
immunoprecipitated (IP) with antibodies to Jak2, Stat1
,
or Stat3, then blotted with anti-phosphotyrosine antibodies
(
-PY). B, Nb2 cells were incubated at 37 °C
for 30 min at low cell density (0.5 × 108 cells/ml;
lanes a and b) or high cell density (4.0 × 108 cells/ml; lanes c-f) before stimulation
with (+) or without (
) 100 nM of ovine PRL for 15 min at
low cell density (lanes a, b, e, and
f) or high cell density (lanes c and
d). Clarified cell lysates were immunoprecipitated with
antibodies to Jak2, Stat1
, Stat3, or Stat5, then blotted with
anti-phosphotyrosine antibodies (
-PY). Parallel samples
of whole cell lysates were also immunoblotted with
-pMAPK
antibodies.
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Fig. 3.
A low molecular weight factor from high
density medium selectively modulates PRL signals. A,
effect of conditioned high density medium on PRL signals.
Quiescent Nb2 cells were incubated at 37 °C for a total of 45 min in
the presence (+) or absence ( ) of 100 nM ovine PRL during
the final 15 min at either low cell density (0.5 × 108 cells/ml; lanes a and b) or high
cell density (4 × 108 cells/ml; lanes c
and d) in control medium (Ctrl), or at low cell
density in conditioned HDM that had been either freshly generated
(Fresh; lanes e and f), stored at
4 °C for 24 h (Old; lanes g and
h), boiled for 10 min and clarified (12,000 × g for 30 min; Boiled; lanes i and
j), or boiled, clarified and dialyzed using a molecular
weight cutoff of 10 kDa against 100 volumes of starvation medium
(Dialyzed; lanes k and l). Cell
lysates were immunoprecipitated (IP) with antibodies to Jak2
or Stat1
, and blotted with anti-phosphotyrosine antibodies
(
-PY). B, effect of low molecular weight
fraction from boiled conditioned high density medium on PRL
signals. Quiescent Nb2 cells were incubated at 37 °C for
a total of 45 min in the presence (+) or absence (
) of 100 nM ovine PRL during the final 15 min at low cell density
(lanes a and b) or high cell density (lanes
c and d) in control medium (Ctrl), or at low
cell density in medium reconstituted with the low molecular weight
fraction (<10 kDa) from conditioned high density medium (Low
MW; lanes e and f) or the corresponding low
molecular weight fraction from control medium (Ctrl Low MW;
lanes g and h). Cell lysates were
immunoprecipitated with antibodies to Jak2 or Stat1
and blotted with
anti-phosphotyrosine antibodies (
-PY). Parallel samples
of whole cell lysates were also immunoblotted with
-pMAPK antibodies
to detect activated ERK1 and ERK2. C, the low molecular
weight factor is insensitive to proteinase K. Control medium
(Ctrl) and conditioned HDM were boiled for 10 min, clarified
(12,000 × g for 30 min at 4 °C), and treated with
(+) or without (
) proteinase K (50 µg/ml) at 37 °C for 1 h,
reboiled for 5 min, and reclarified by centrifugation at 12,000 × g for 30 min at 4 °C. The resulting four supernatants
then served as incubation medium to examine the sensitivity of the low
molecular weight factor to proteinase K. Nb2 cells were incubated at
37 °C for a total of 45 min in the presence (+) or absence (
) of
100 nM ovine PRL during the final 15 min in media
representing either control or conditioned HDM that had been treated
with (+) or without (
) proteinase K as indicated. Clarified cell
lysates were immunoprecipitated (IP) with antibodies to Jak2
or Stat1
, followed by blotting with anti-phosphotyrosine antibodies
(
-PY). D, proteinase K is enzymatically active
under the conditions used. Condi tioned low-density medium was spiked with prolactin (10 nM) and incubated for 30 min at 37 °C in the presence or
absence of proteinase K before cells were added for a 5-min stimulation
period and Jak2 tyrosine phosphorylation was assayed.
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Fig. 4.
Modulation of discrete PRL signals in Nb2
cells at high cell density is mimicked by staurosporine, a potent
protein kinase inhibitor. A, effect of calcium
ionophore or cyclic nucleotide mimetics on PRL signals. Nb2 cells were
incubated at 37 °C for a total of 45 min with control medium alone
(Ctrl), A23187 (1 µM), 8-Br-cAMP (1 mM), 8-Br-cGMP (1 mM), or
isobutylmethylxanthine (MIX; 100 µM) in the
presence (+) or absence ( ) of 100 nM ovine PRL during the
final 15 min. Cell lysates were immunoprecipitated (IP) with
antibodies to Jak2, Stat1
, or Stat3, and blotted with
anti-phosphotyrosine antibodies (
-PY). B,
effect of protein kinase modulators on PRL signals. Nb2 cells were
incubated at 37 °C for a total of 45 min with control medium alone
(Ctrl), phorbol 12-myristate 13-acetate (PMA; 1 µM), staurosporine (Stsp; 500 nM),
bis-indolelylmaleimide (BIM; 500 µM), or
genistein (Gstn; 200 µM) in the presence (+)
or absence (
) of 100 nM ovine PRL during the final 15 min. Cell lysates were immunoprecipitated (IP) with
antibodies to Jak2, Stat1
, or Stat3, and blotted with
anti-phosphotyrosine antibodies (
-PY). C,
effect of staurosporine or genistein on PRL-induced Stat5 signals.
Quiescent Nb2 cells were incubated at 37 °C with staurosporine
(Stsp; 500 nM) or genistein (Gstn;
200 µM) for a total of 45 min with (+) or without (
)
100 nM ovine PRL during the final 15 min. Cell lysates were
immunoprecipitated (IP) with antibodies to Jak2, Stat1
,
Stat3, Stat5a, or Stat5b, and blotted with anti-phosphotyrosine
antibodies (
-PY).
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Fig. 5.
A low molecular weight factor from high
density medium inhibits cell growth. A, effect of
conditioned high density medium on cell growth. Nb2 cells were cultured
in control medium (Ctrl) or in conditioned HDM at 37 °C
for 48 h, and cell numbers were assessed by the MTT assay. MTT
formation is expressed as absorbance (OD) at 590 nm. Error
bars indicate the mean ± S.E. (n = 3).
Differences between groups were compared by Student's t
test (***, p < 0.0001). B,
concentration-dependent growth inhibition by partially
purified low molecular weight factor derived from boiled clarified
conditioned high density medium. Nb2 cells were cultured for 48 h
in the presence of increasing concentrations of the partially purified
factor derived from the low molecular weight fraction after dialysis of
boiled and clarified conditioned HDM. The concentration of the
partially purified inhibitory factor is presented as units relative to
its concentration in conditioned HDM, i.e. the unit 1.00 represents 1× HDM. Cell numbers were assayed by the MTT method, and
the data are presented as percent growth inhibition relative to cells
treated with corresponding fractions derived from dialysis of boiled
and clarified control culture medium. Error bars indicate the mean ± S.E. (n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, interferons, glial maturation
factor-
, and mammastatin (for review, see Ref. 37). We have
described the partial purification of a heat-stable low molecular
weight inhibitory activity from conditioned high cell density culture medium. The activity was also resistant to extended treatment with the
nonselective endoprotease, proteinase K. It is therefore possible that
the factor is not a peptide. Proteinase K is active over a wide pH
range, is insensitive to metal ions, and cleaves peptide bonds
nonselectively after nonpolar or aromatic amino acids (28), which
account for more than half of the natural amino acids. It will now be
important to determine whether the endogenous inhibitor is indeed of
nonpeptidyl nature, or whether it is a peptide without proteinase
K-cleavable amide bonds. If the factor were not to be a peptide,
membrane lipid derivatives such as prostaglandins or leukotrienes
represent one candidate class of molecules, because eicosanoids can
inhibit cell growth (38), are quickly released, and activate specific
receptors (39). Further chemical analysis and purification of this
factor is being pursued.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1 DK52013-01A1 and Uniformed Services University of the Health Sciences Grant RO74HF.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
Uniformed Services University of the Health Sciences School of
Medicine, Bethesda, MD 20814. Tel.: 301-295-3801; Fax: 301-295-1640; E-mail hrui{at}usuhs.mil.
2 H. Yamashita, J. Xu, and H. Rui, unpublished data.
3 H. Yamashita and H. Rui, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PRL, prolactin; MAPK, mitogenactivated protein kinase; HDM, high density medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PKC, protein kinase C.
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