Role of Cyclophilin B in Prolactin Signal Transduction and Nuclear Retrotranslocation
Michael A. Rycyzyn,
Sean C. Reilly,
Kerri OMalley and
Charles V. Clevenger
Department of Pathology and Laboratory Medicine University of
Pennsylvania Medical Center Philadelphia, Pennsylvania 19104
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ABSTRACT
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The pleiotropic actions of PRL are necessary for
mammary growth and differentiation and in vitro lymphoid
proliferation. The proximal action of this ligand is mediated by its
cell surface receptor via associated networks. PRL action, however, is
also associated with the internalization and translocation of this
hormone into the nucleus. To delineate the mechanism of this
retrotranslocation, a yeast two-hybrid screen was performed and
revealed an interaction between PRL and cyclophilin B (CypB). CypB is a
peptidyl prolyl isomerase (PPI) found in the endoplasmic
reticulum, extracellular space, and nucleus. The interaction
between CypB and PRL was subsequently confirmed in vitro
and in vivo through the use of recombinant proteins and
coimmunoprecipitation studies. The exogenous addition of CypB
potentiated the 3H-thymidine incorporation of
PRL-dependent cell lines up to 18-fold. CypB by itself was nonmitogenic
and did not potentiate the action of GH or other interleukins. CypB did
not alter the affinity of the PRL receptor (PRLr) for its ligand, or
increase the phosphorylation of PRLr-associated Jak2 or Stat5a. The
potentiation of PRL-action by CypB, however, was accompanied by a
dramatic increase in the nuclear retrotranslocation of PRL. A CypB
mutant, termed CypB-NT, was generated that lacked the wild-type
N-terminal nuclear localization sequence. Although CypB-NT demonstrated
levels of PRL binding and PPI activity equivalent to wild-type CypB, it
was incapable of mediating the nuclear retrotranslocation of PRL or
enhancing PRL-driven proliferation. These studies reveal CypB as an
important chaperone facilitating the nuclear retrotransport and action
of the lactogenic hormones.
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INTRODUCTION
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The functions of the somatolactogenic hormone PRL are mediated by
its receptor (PRLr), a member of the superfamily of type I cytokine
receptors (1, 2, 3). Ligand-induced dimerization of these receptors
activates several associated signaling cascades including the Jak-Stat,
Ras-Raf, and Fyn-Vav pathways (4, 5, 6, 7). None of these signaling pathways
is uniquely associated with the receptor for PRL; indeed, many other
type I receptors utilize these signaling pathways (8). Thus, the
mechanisms through which specific patterns of PRL-induced gene
expression and action are induced or potentiated have remained
elusive.
The activation of PRL receptor-affiliated signaling pathways is also
associated with the internalization of the ligand (9). PRL is
internalized via an endosomal-like pathway and transported across the
endoplasmic reticulum (ER) and nuclear envelopes, a process
termed nuclear retrotranslocation (10, 11). The retrotranslocation of
PRL appears to be an active process, requiring costimulation by
additional ligand, such as interleukin-2 in lymphocytes and epidermal
growth factor in breast epithelium (10). The retrotranslocation of PRL
is of functional consequence, as nuclear PRL provides a necessary
comitogenic stimulus for IL2-driven growth (12, 13). The phenomenon of
reverse protein transport was characterized initially through the study
of retrotranslocated bacterial and viral proteins, and peptides
destined for presentation on the major histocompatibility complex
(14, 15, 16, 17). These studies have revealed that protein retrotranslocation
is dependent upon the retrograde transport through the
protein-conducting channel formed by the Sec61 complex in the ER
membrane. The retrotranslocation of peptide hormones also appears to be
widespread as several peptide hormones, such as GH, epidermal growth
factor (EGF), nerve growth factor (NGF), platelet-derived growth factor
(PDGF), fibroblast growth factor (FGF), insulin, and interleukin-5,
have been noted within the nucleus (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). The mechanism of such
retrotranslocation and the nuclear action of these peptide hormones,
however, have remained uncertain.
We reasoned that if PRL acts directly within the nucleus, it must do so
through a binding partner, or chaperone, as this hormone lacks either
an enzymatic activity or a nuclear translocation signal sequence. We
demonstrate here that cyclophilin B (CypB), initially identified by
yeast two-hybrid analysis, binds to PRL both in vitro and
in vivo. Our findings reveal that CypB significantly
potentiates the interrelated phenomena of nuclear retrotranslocation
and PRL action.
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RESULTS
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The Interaction of PRL with CypB
To identify potential binding partners of PRL, a yeast two-hybrid
screening strategy was employed. A Jurkat cDNA library fused to the
activation domain of GAL1 was screened against a "bait"
of a chimeric lexA-PRL construct expressed in the yeast EGY48 cell
line. Sequencing of the cDNA obtained from yeast clones demonstrating
both ß-galactosidase activity and positive growth on selective medium
was performed, revealing that fully 10% of the interacting clones
contained a full-length human CypB cDNA. As shown in Table 1
, the interaction between PRL and CypB
in yeast was specific, as determined by several positive and negative
controls.
To confirm that CypB directly interacts with PRL, coimmunoprecipitation
studies using recombinant CypB and purified PRL were performed on the
admixed proteins (Figs. 1A
and Fig. 3E
).
These studies revealed that a direct interaction between the purified
proteins occurred in vitro. Furthermore, the data revealed
that the introduction of either reducing agent or divalent calcium
facilitated the direct interaction of CypB with PRL. The facilitation
of CypB and PRL binding was ion specific as the addition of equivalent
concentrations of divalent magnesium did not alter this interaction
(not shown). In the presence of calcium, approximately 32% (±2%,
n = 3) of PRL bound to CypB when mixed at equimolar ratios (Fig. 3E
). The addition of cyclosporin A (CsA), at a therapeutic
concentration, also enhanced this interaction. These data would suggest
that PRL does not interact with the peptidyl prolyl isomerase (PPI)
pocket in CypB that engages CsA. Indeed, preliminary data using
GST-CypB chimera would indicate that PRL binds to the C terminus of
CypB (not shown). This interaction appears specific, in that a
recombinant form of the highly homologous cyclophilin family member
CypA failed to interact with PRL (Fig. 1D
). Further confirmation of the
CypB-PRL interaction in vivo was obtained by the direct
coimmunoprecipitation of PRL with CypB from human serum (Fig. 1E
).
Analysis of anti-CypB immunoprecipitates from normal human serum with a
PRL immunoassay has revealed that approximately 12.5% (± 1.1%,
n = 3) of circulating PRL is engaged to CypB.

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Figure 1. CypB Interacts with and Modulates PRL-Driven
Proliferation
A, In vitro binding assays indicate PRL
associates with CypB. After admixture of these proteins (10 ng of
each/200 µl) under various binding conditions indicated above,
anti-PRL immunoprecipitates were subject to immunoblot analysis with
anti-(V5)CypB. One of three representative experiments is presented.
Where indicated, molecular mass is in daltons. Quantitation of these
immunoblots was performed by scanning densitometry.
Coimmunoprecipitation of the admixed proteins with anti-(his)CypB,
followed by immunoblotting with anti-PRL yielded similar results (not
shown). B, Effect of CypB on PRL-driven proliferation at 48 h. Nb2
cells were incubated with varying concentrations of PRL and CypB or
CypB alone without refeeding, and proliferation was evaluated by the
addition of 0.5 µCi of 3H-thymidine. Y-axis
values represent ratio of 3H-thymidine
incorporation of Nb2 cells stimulated with PRL and
CypB/3H-thymidine incorporation of Nb2 cells
receiving PRL alone (n = 3, the SEM was
<10% of each of the means) One of five representative experiments is
shown. Mean absolute 3H-thymidine incorporation
observed in the resting Nb2 cells used here was 6.4 x
101 cpm; for the Nb2 cells stimulated with 50
pM of PRL alone, incorporation was 1.9 x
103 cpm. C, Effect of refeeding on CypB
enhancement of PRL-driven proliferation. Nb2 cells were incubated with
250 pM PRL and varying concentrations of CypB,
and after 48 h the cultures were fed with 100 µl of DMEM
containing ITS+. Proliferation was assayed at 72 h as above while
absolute cell number and cell viability were ascertained by
hemocytometry and trypan blue exclusion. All Y-axis values are
presented as fold increase over PRL alone (n = 3,
SEM was <10% of each of the means). Mean
absolute 3H-thymidine incorporation observed in
cells stimulated with 250 pM PRL alone was
5.4 x 102; mean viable cell number with PRL
alone was 6.6 x 104; viable cells receiving
250 pM PRL alone was 38%, PRL + 500
nM CypB was 83%. One of two representative
experiments is shown. D, In vitro binding assays reveal the
related cyclophilin family member CypA does not interact with PRL.
After admixture of either CypA or CypB with PRL in binding buffer
containing 5 mM CaCl2,
anti-PRL immunoprecipitates were subject to immunoblot analysis with
anti-V5(Cyp). One of two representative experiments is presented. E,
PRL associates with CypB in vivo. Normal human serum was
immunoprecipitated with either anti-PRL or anti-CypB and subjected to
SDS-PAGE and immunoblot analysis with the corresponding antibody.
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Figure 3. Retrotranslocation of the PRL/CypB complex
Panel A, Effect of CypB and CypB-NT on PRL internalization and nuclear
retrotranslocation as assessed by immunofluorescence microscopy. T47D
human breast cancer cells were incubated for 4 h with 10 ng EGF/ml
in the presence of (a) no exogenous addition, (b) 10 ng PRL/ml, (c) 10
ng PRL/ml and 100 ng CypB/ml, or (d) 10 ng PRL/ml and 100 ng
CypB-NT/ml. The cells were then labeled with anti-PRL indirect
immunofluorescence (magnification, 350x). At least 200 cells were
evaluated in each experiment; one of three representative experiments
is shown. Panel B, Effect of CypB and CypB-NT on PRL internalization
and nuclear retrotranslocation as assessed by double label confocal
microscopy. T47D cells were stimulated with 10 ng EGF/ml and 10 ng/ml
PRL in the presence of CypB (panels a and c) or CypB-NT (panels b and
d) for 4 h. The cells were then labeled with anti-PRL indirect
immunofluorescence and propidium iodide. Confocal microscopy of the
anti-PRL (panels a and b) and DNA (panels c and d) signals revealed
colocalization of a fraction of the internalized PRL within the nucleus
in the presence of CypB, but not CypB-NT (magnification, 600x). Panel
C, Biochemical assessment of the internalization of the PRL/CypB
complex. Nb2 cells were cultured in the presence of PRL and V5-tagged
CypB. After harvest at the indicated times, washed cell lysates were
immunoprecipitated with antiserum against PRL and subjected to SDS-PAGE
and immunoblot analysis with an anti-V5 antibody. One of two
representative experiments is shown. Panel D, Biochemical evaluation of
the nuclear retrotransport of CypB. Nb2 cells were cultured in the
presence of PRL and V5-tagged CypB for various time intervals. After
harvesting and washing, nuclear ("N") and cytosolic extracts
("C") were subjected to SDS-PAGE and immunoblot analysis with
anti-V5 antibody. One of two representative experiments is presented.
Panel E, Biochemical quantitation of the PRL/CypB complex in
vitro. V5/His-tagged PRL was mixed with either GST or GST-CypB
coupled to glutathione-Sepharose beads. Bead flow through
(i.e. containing PRL not bound to beads) was
immunoprecipitated with anti-His antibody. PRL bound to the GST or
GST-CypB beads (indicated by bound) and the anti-His PRL
immunoprecipitates from the bead flow through (indicated by flow
through) were subject to immunoblot analysis with anti-V5-HRP. The
results presented here used 50 ng each of PRL and CypB
(i.e. near equimolar proportions of each), and similar
results in terms of percent binding were obtained with 100 ng of each
protein. Representative of one of two experiments. Quantitation was
performed by scanning densitometry. Panel F, Biochemical assessment of
the intracellular localization of endogenous CypB in Nb2 and T47D
cells. Resting Nb2 (7.5 x 105) and T47D (5.0 x
104) cells were stimulated for 2 h with PRL (Nb2) or
PRL/EGF (T47D). Cytoplasmic ("C") and nuclear ("N") extracts
were subjected to immunoblot analysis with anti-CypB.
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Functional Consequences of the CypB-PRL Interaction
The effect of exogenous CypB on PRL-driven proliferation was
examined with the rat Nb2 T-cell and the human T47D breast cancer
lines. In response to exogenous PRL, Nb2 cells demonstrate
dose-dependent proliferation (30, 31). The addition of CypB into the
PRL-containing Nb2 medium, cultured without refeeding for 48 h,
resulted in up to a 9-fold enhancement of
3H-thymidine incorporation into DNA in comparison
to Nb2 cultures that received only PRL (Fig. 1B
). Indeed, this
dose-dependent, biphasic effect was most prominent at physiological
concentrations (510 nM) of CypB and PRL (500
pM) found in human serum. The CypB enhancement of
PRL-driven growth was identical, regardless of whether or not CypB and
PRL were first premixed, and similar proliferative responses were
observed with the T47D line (data not shown). In the absence of ligand,
CypB at any concentration was neither mitogenic for, nor inherently
toxic to, the Nb2 cells. To address whether the biphasic curve observed
was due to the cells exhausting the medium of nutrients as a
consequence of growth enhanced by CypB, Nb2 cultures receiving 250
pM PRL in addition to CypB were fed at 48 h with 100
µl of defined medium and assayed at 72 h. This refeeding
abolished the previously observed biphasic curve (Fig. 1C
). Concomitant
with the increase in 3H-thymidine incorporation,
further analysis indicated a dose-dependent, 4.5-fold increase in
viable cell number. To determine the effect of CypB on cellular
proliferation driven by the larger family of cytokines, to which PRL
belongs, the incorporation of 3H-thymidine driven
by interleukin-2 (IL-2), interleukin (IL-3), human GH (hGH), and
recombinant rat GH (rGH) and bovine GH (bGH) was examined (data not
shown). Neither IL-2- nor IL-3-driven
3H-thymidine incorporation was altered by the
addition of varying concentrations of CypB. While CypB was found to
interact with hGH and potentiate hGH effects on Nb2 cells, no
enhancement of nonprimate GH-driven 3H-thymidine
incorporation was observed. However, we have not entirely ruled out
CypB may potentiate other actions of GH, not assessed by Nb2 bioassay.
Taken together, these data would argue that PRL action is significantly
potentiated, in a synergistic manner, by physiological concentrations
of CypB.
CypB Potentiation of PRL Action Is Not Mediated by the Modulation
of either Receptor-Associated Ligand Affinity or Signal
Transduction
A mechanism through which CypB could potentiate PRL action is its
interaction and modulation of the function of the PRL/PRLr complex.
Indeed, evidence for other transmembrane receptors has demonstrated
that the interaction of cyclophilins with either the extracellular or
intracellular domains can modulate receptor transduction (32). Thus, it
was conceivable that CypB either enhanced the interaction of PRL with
the PRLr, or in some other manner enhanced the signaling output of the
PRLr complex. To test the former hypothesis, radioligand binding
studies using 125I-PRL on Nb2 cells in the
presence or absence of CypB, followed by Scatchard analysis, was
performed. These data revealed no alteration in either the affinity
(Kd = 2 x 10-9
M) or number of the PRLr present at the surface of Nb2
cells. The latter hypothesis, i.e. alteration of the
PRLr-associated signaling output, was evaluated by analysis of
PRL-induced Jak2 and Stat5a activation by ligand in the presence and
absence of CypB (Fig. 2
). No demonstrable
alteration in PRL-stimulated Jak2 or Stat5a phosphorylation was
observed over multiple time points. Taken together, these data would
argue against CypB-induced alterations of either PRL-PRLr interaction
or PRLr-associated signaling.

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Figure 2. PRL-Induced Tyrosine Phosphorylation of Jak2 and
Stat5a in the Presence of CypB
Resting Nb2 cells were treated with PRL, CypB, or a combination of the
two. Cell lysates were immunoprecipitated with antiserum against Jak2
(A) or Stat5a (B), and precipitated proteins were analyzed for
phosphotyrosine content. One of two representative experiments is
shown.
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CypB Enhances the Nuclear Retrotranslocation of Exogenous PRL
The hypothesis that CypB could enhance the nuclear
retrotranslocation of PRL, and thereby PRL-driven proliferation,
was examined by standard anti-PRL immunofluorescence microscopy
(Fig. 3A
) and by double-label
laser-scanning confocal microscopy. As previously documented (33),
indirect immunofluorescence of T47D human breast cancer cells labeled
with an anti-PRL antibody demonstrates a diffuse, speckled pattern of
cytosolic immunofluorescence in the majority of cells, consistent with
internalization of PRL into endosome-like vesicles. However, precedent
data in the L2 cloned T-cell line had indicated that a comitogenic
stimulus was required before appreciable nuclear retrotranslocation of
PRL was detectable (12, 13). Indeed, inclusion of the comitogen EGF
into the defined T47D culture medium induced demonstrable anti-PRL
immunofluorescence over approximately 10% of the T47D nuclei (Fig. 3A
, panel b). The nuclear retrotranslocation of PRL, however, was
significantly enhanced by the inclusion of CypB into this defined
medium (Fig. 3A
, panel c), resulting in 95100% of the T47D nuclei
demonstrating anti-PRL immunofluorescence within 24 h after addition
of PRL and CypB. These data were secondarily confirmed in both T47D
(Fig. 3B
) and Nb2 (not shown) cells labeled with anti-PRL
immunofluorescence and the DNA intercalating agent propidium iodide,
when analyzed by confocal microscopy. In both cell types, the addition
of CypB resulted in a visible anti-PRL immunofluorescent signal in the
nuclear plane. These data were also confirmed biochemically at two
levels. First, the intracellular internalization of a PRL/CypB complex
was studied by PRL/CypB coimmunoprecipitation analysis of lysates
obtained from Nb2 cells incubated in the presence of exogenous PRL and
V5 epitope-tagged CypB as a function of time (Fig. 3C
). These data
demonstrated that the extracellular complexes formed between CypB and
PRL are internalized intracellularly, presumably via PRLr-mediated
endocytosis of the complex. Second, the nuclear retrotransport of
exogenous, epitope-tagged CypB was confirmed biochemically by
anti-V5/CypB immunoblot analysis of nuclear and cytosolic cellular
fractions (Fig. 3D
). These data demonstrated that extracellular CypB
was rapidly internalized within 30 min after exogenous addition,
paralleling the internalization of the PRL/CypB complex. These findings
also showed that neither PRL nor CypB underwent significant alteration
of their molecular mass after internalization, excluding proteolytic
processing as a mechanism involved in the retrotransport of the
PRL/CypB complex. This process appeared to be dependent upon cell
surface expression of the PRLr, as cells lacking PRLr expression, such
as IL-3-dependent 32D line, failed to demonstrate the internalization
of either PRL or CypB (data not shown). Further confirmation of the
ability of endogenous CypB (i.e. that synthesized by the
cell itself) to "shuttle" into the nucleus was obtained by
anti-CypB immunoblot analysis of nuclear and cytosolic cellular
fractions generated from resting and PRL-stimulated Nb2 and T47D cells
cultured in defined medium (Fig. 3F
). These data revealed that
endogenously synthesized CypB was capable of nuclear entry in the
presence or absence of PRL, confirming prior studies demonstrating that
up to 50% of endogenous CypB can be found within the nucleus in
resting or mitogen-stimulated cells (34). Collectively, these data
would indicate that CypB, as a "reverse chaperone," facilitates the
retrotranslocation of PRL into the nucleus.
Mechanism of CypB-Mediated Nuclear Retrotranslocation of PRL
Examination of the amino acid sequence of CypB revealed a putative
nuclear translocation signal in its amino terminus (Fig. 4A
). The role of this sequence in the
CypB-mediated retrotranslocation of PRL, and its associated enhancement
of 3H-thymidine incorporation, was tested by
mutagenesis approaches. A deletion construct of CypB, termed CypB-NT,
was generated by conventional PCR-based mutagenesis and expressed in
Drosophila S2 cells. After purification, comparison
between the wild-type CypB and CypB-NT revealed comparable levels of
PPI activity (not shown), confirming that the mutant construct was
appropriately folded and bioactive. Further evaluation of the CypB-NT
mutant revealed that its interaction with PRL was not affected by
deletion of the N terminus (Fig. 4B
). Given this confirmatory data, the
function of the CypB-NT mutant was tested in both T47D and Nb2 cells.
The inclusion of CypB-NT into the culture medium resulted in an absence
of detectable anti-PRL immunofluorescence in any nucleus (Fig. 3
, A and
B) with a dose-dependent loss of enhanced PRL-driven
3H-thymidine incorporation (Fig. 4C
). These data
establish a role for the N terminus of CypB in the nuclear
retrotransport of PRL and confirm the growth-enhancing properties of
nuclear PRL.

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Figure 4. Deletion of N-Terminal Nuclear Translocation
Sequence in CypB Blocks the CypB-Mediated Enhancement of PRL-Driven
Proliferation
A, Comparison of N-terminal regions of CypA and CypB with the nuclear
localization sequence of the SV40 large T antigen. B, In
vitro interaction between PRL and CypB or CypB-NT was assessed,
revealing that the deletion of the N terminus of CypB does not
alter its interaction with PRL. C, CypB-NT competitively inhibits
PRL/CypB-driven proliferation. Nb2 cells were stimulated with 500
pM PRL ± 50 nM CypB ± 5
to 500 nM CypB-NT (n = 3, the sem was <10% of
each of the means). Mean absolute 3H-thymidine
incorporation observed in the resting Nb2 cells used here was 6.3
x 101 cpm; for the Nb2 cells stimulated with 500
pM of PRL alone, incorporation was 6.8 x
102 cpm.
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DISCUSSION
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We have demonstrated that the nuclear retrotranslocation of the
somatolactogenic hormone PRL is mediated by its direct interaction with
CypB. Indeed, these findings confirm that the nuclear
retrotranslocation of somatolactogenic hormones enhances cell
proliferation, an effect ablated by the deletion of the nuclear
translocation signal in CypB. CypB is a member of the larger
cyclophilin family of cis-trans PPI (35, 36, 37). This family of
proteins was initially identified in the search for binding partners
for the extensively used immunosuppressive agent CsA. CsA interacts
with the cyclophilins with high affinity, inhibiting their PPI activity
and also inhibiting the action of the phosphatase calcineurin,
necessary for NF/AT-transactivated expression of IL-2 (38, 39, 40, 41, 42, 43). Indeed,
the effect of CsA on CypB-intrinsic PPI activity is the most pronounced
within the cyclophilin family.
Despite its relationship to the cyclophilin family, the physiological
function of CypB had remained uncertain. Cyclophilins regulate the
activity of transduction cascades, as evidenced by their modulation of
transforming growth factor-ß (TGF-ß) signaling and the
transactivation of c-myb (32, 44). Indeed, prior data
suggested that the interaction and function of the PRL-PRLr complex
were CsA sensitive (45, 46). In this capacity, recent data indicate
that the cyclophilins may mediate these effects through their action as
protein chaperones. Cyclophilins, via their PPI activity, facilitate
protein folding and have been shown to contribute to the maturation of
several proteins, including carbonic anhydrase and the human
immunodeficiency virus (HIV) glycoprotein Gag (47, 48). While
cyclophilin-mediated protein maturation may occur on both sides of the
ER membrane, existing data strongly suggest that CypB associates with
nascent peptides during their transport across the ER membrane. Indeed,
a CsA-sensitive protein of the molecular mass of CypB associates with
nascent pre-PRL and the Sec61 transporter apparatus (49). While some
studies have suggested the presence of a widespread, but as yet
unidentified, cell-surface receptor specific for CypB (61), our
analysis of the cell lines studied here provide no evidence for this
claim. Indeed, one interpretation of the findings presented here is
that the mechanism for extracellular CypB action and internalization is
through its interaction with the PRL-PRLr complex.
Additional clues of CypB function can be obtained through the
structural analysis of this protein. CypB is a ß-barrel protein
containing an N-terminal ER-leader and C-terminal ER-retention
sequences (50, 51). CypB has been observed in the ER and nucleus and
can be found in appreciable levels in blood (150 ng/ml) and breast milk
(52, 53). Proteolysis of the C-terminal ER retention motif results in
the secretion of CypB (51); however, the mechanism for its nuclear
localization had not been previously questioned. Clearly, the
observations presented here indicate that the N terminus of the mature
CypB contains a localization signal that mediates its nuclear
translocation. Taken together, these functional and localization data
indicate that CypB could serve as chaperone mediating the transport
and/or function of associated proteins. On the basis of the results
presented here, we hypothesize that the extracellular CypB/PRL complex
is internalized during PRL/PRLr endocytosis. Given the putative ability
of CypB to associate with the Sec61 complex and our observation of CypB
internalized within the cytosol and nucleus, we speculate that the
CypB/PRL complex is capable of retrotransport across the ER membrane
through the Sec61 transport apparatus. Upon gaining entry to the
cytosol, the N-terminal nuclear localization sequence of CypB is
necessary for the nuclear retrotransport of PRL. Clearly, the
mechanisms of CypB retrotransport across the ER membrane, and the
ability of this pathway to distinguish between nascent and exogenous
ligand, are active areas of investigation within our laboratory. We
currently speculate that such distinctions may be made by the cell on
the basis of posttranslational modifications to CypB, and
coimmunoprecipitation studies of exogenously added epitope-tagged
proteins and Sec61 should serve to clarify this.
CypB stimulates PRL-driven proliferation in a dose-dependent manner.
The biphasic nature of this response at 48 h is similar to that
seen for the dose-response curves of both PRL and GH (54, 55). However,
it appears that this biphasic response observed at 48 h is the
consequence of rapid cell growth removing the defined medium of
essential nutrients. Indeed, when fed at 48 h and assayed at
72 h, this biphasic response is no longer observed. This coupled
with the 4.5-fold increase in viable cell number at increasing doses of
CypB would suggest the PRL/CypB complex transported to the nucleus is
providing a secondary signal that is enhancing the proliferative
ability/survivability of these cells.
While several laboratories have identified both PRL and GH in the
nucleus (13, 18, 56), not all investigators have been able to confirm
these findings (33). The one group not able to confirm the
retrotranslocation of somatolactogenic hormones, however, failed to
utilize necessary costimulatory factors, such as IL-2 and EGF, in their
assays. Indeed, cross-talk between the EGF receptor and members of the
somatolactogenic family appears to regulate the downstream signaling
activity of each (57, 58, 59). We are examining whether these costimulatory
factors may serve to enhance the expression or secretion of CypB,
thereby facilitating the nuclear retrotransport of PRL.
Previous data have indicated that the action of PRL may be mimicked by
dimerizing anti-PRLr antibodies (60). While such antibody-mediated PRLr
dimerization could induce the proliferation of responsive cell lines,
it occurred with significant reductions in efficacy, and with potencies
70- to 4700-fold less than that observed for ligand. One interpretation
of these findings was that the anti-PRLr antibodies used were unable to
optimally orient the dimerized PRLr complex for maximal signaling. In
light of the data obtained here, however, we hypothesize that in the
absence of ligand (or specifically, an intranuclear PRL/CypB complex)
maximal potentiation of receptor-driven proliferation by dimerizing
anti-PRLr antibodies could not occur.
The findings presented here mechanistically provide a basis for the
functional transport and activity of the somatolactogenic hormones
within the nucleus. This action may be mediated by the direct
interaction of the PRL-CypB complex with transcription factors. Earlier
data have demonstrated that members of the immunophilin family directly
interact and modulate the activity of transcription factors (44, 64).
However, interaction of the PRL-CypB complex with transcription factors
may not be exclusively mediated by CypB alone. Indeed, the second most
common class of proteins interacting with PRL in our two-hybrid screen
were transcription factors, an initial finding now confirmed by
coimmunoprecipitation studies (M. A. Rycyzyn and C. V.
Clevenger, manuscript in preparation). These findings suggest that
intranuclear PRL acts as a protein scaffold, targeting the PPI activity
of CypB to specific transcription factors. Thus, the direct
intranuclear interactions of the peptide hormones may facilitate or
enhance their ligand-associated actions. Given the important
physiological and pathological functions of PRL, modulation of the
function of such intranuclear CypB-ligand complexes, as
demonstrated here with the CypB-NT mutant, could be of appreciable
therapeutic utility.
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MATERIALS AND METHODS
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Yeast Two-Hybrid Analysis
Full-length cDNA for human PRL was generated by PCR using
specific primers containing BamHI sites:
5'-CGGGATCCCCTTGCCCATCTGTCCGGCGGGGCT-3' and
5'-CGGGATCCTTAGCAGTTGTTGTTGTGGAT-3', cloned into the yeast vector
pJK202 and cotransfected into the yeast strain EGY48 along with a
Jurkat cDNA library inserted into the pJG4 vector. Selection of
positive interacting clones was performed as previously described
(61).
Generation and Expression of Recombinant Proteins
Full-length CypB generated by PCR from the insert isolated in
the yeast two-hybrid screen using specific primers containing
KpnI and XhoI sites
5'-CGGGTACCACCATGATGAAGGTGCTCCTTGCCGCCGCC-3' and
5'-CGCTCGAGCTCCTTGGCGATGCCAAAGGG-3', with the forward primer containing
a Kozak signal sequence. Full-length cDNA for human CypA was generated
from Jurkat cells by RT-PCR using an oligo-dT synthesized cDNA pool and
specific primers containing KpnI and XhoI sites
5'-CGGGTACCACCATGATGGTTAACCCCACCGTG-3' and
5'-CGCTCGAGTTCGAGTTGTCCACAGTC-3', with the forward primer
containing a Kozak signal sequence. Full-length PRL cDNA was generated
by PCR using a specific forward primer containing a KpnI
site and Kozak signal sequence:
5'-CGGGTACCACCATGATGAACATCAAAGGATCGCCATGGAAAGGG-3'; and a reverse
primer containing a XhoI site and a myc-tag with
two stop codons immediately after the tag:
5'-CGCTCGAGTTACTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGCAGTTGTTGTTGTGGATGAT-3'.
CypB-NT was generated by overlapping PCR mutagenesis. The forward CypB
primer containing the KpnI site and Kozak signal sequence
was combined with the reverse primer
5'-AAAATACACCTTGGCCGCAGAAGGTCCCGG-3', while the reverse primer
containing the XhoI site was combined with the
forward primer
5'-GCGGCCAAGGTGTATTTTGACCTACGAATTGGA-3'. The resulting PCR
products were purified, mixed, and reamplified with the forward and
reverse CypB primers. The resulting PCR product, lacking amino acid
residues 212 of the mature peptide while retaining the leader
sequence, was confirmed by dideoxynucleotide sequencing. Each of the
above (PRL, CypA, CypB, and CypB-NT) PCR products were purified,
digested, and subcloned into pAc5/V5-HisA of the Drosophila
Expression System (Invitrogen, San Diego, CA), containing
a bifunctional V5/His-tag. The sequences of all inserts were confirmed
by dideoxynucleotide sequencing. Nineteen micrograms of the vector
containing one of the above constructs were cotransfected with 1 µg
of pCOHYGRO into 4 x 106 Drosophila
S2 cells by the CaCl2 method and transfectants
were selected with hygromycin-B according to the manufacturers
instructions (Invitrogen). Myc-tagged PRL was expressed
and secreted into the culture supernatant at levels upwards of 10
mg/liter. This protein was determined to be functionally bioactive by
Nb2 bioassays. His-tagged CypB, CypA, and CypB-NT were expressed
intracellularly and purified as follows: 20 ml cultures containing
approximately 2 x 108 were shaken overnight
at room temperature at 100 rpm, pelleted, and lysed in a minimal NP-40
lysis buffer (50 mM Tris, pH 7.8, 150
mM NaCl, 1% NP-40). Lysates (1 ml) were
clarified and incubated for 30 min with 200 µl of TALON metal
affinity resin (CLONTECH Laboratories, Inc., Palo Alto,
CA) at room temperature. Resin was washed four to five times with wash
buffer (20 mM Tris-HCl, pH 8.0, 100
mM NaCl, 10 mM imidazole)
before being suspended in elution buffer (wash buffer containing 50
mM imidazole). Eluted protein was dialyzed
overnight at 4 C against 5 mM HEPES, pH 8.0, and
quantitated by spectrophotometric technique. All recombinantly
expressed cyclophilins were determined to be fully bioactive by a
standard PPI assay (62). Three different preparations of PRL were used
in the studies presented here: human pituitary isolated PRL (National
Hormone and Pituitary Program, NIDDK); recombinant human PRL from
E. coli (Genzyme Corp., Cambridge, MA); or the
above detailed epitope-tagged human PRL from S2 cells. Although the
S2-derived tagged PRL was used predominantly for the in
vitro binding assays, and the E. coli-derived PRL for
bioassay, each preparation was cross-tested to ensure reproducible
results across the analytic methods used.
In Vitro Binding Assays and Immunoprecipitations
Purified V5/His-tagged CypB or CypA and myc-tagged PRL (10 ng of
each) were admixed in 200 µl binding buffer (10 mM Tris,
pH 7.6, 125 mM NaCl, 10% glycerol) in the presence or
absence 5 mM CaCl2, 1.5
mM 2-mercaptoethanol, or 50 nM CsA for 3 h
at room temperature. Complexes were immunoprecipitated by the addition
of 1 µl polyclonal anti-PRL for 1 h followed by incubation for
1 h with 50 µl Protein-A beads (Life Technologies, Inc., Gaithersburg, MD). Anti-PRL immunoprecipitates were
analyzed by immunoblot analysis with a monoclonal anti-V5-HRP antibody
(1:1000, Invitrogen). Detection of all antigen-antibody
complexes was accomplished by incubation with ECL Plus (Amersham Pharmacia Biotech, Arlington Heights, IL) and exposure to Biomax
film (Eastman Kodak Co., Rochester, NY). For in
vivo coimmunoprecipitation studies, human serum was extensively
precleared with protein-A Sepharose before overnight
immunoprecipitation with 1 µg of either polyclonal anti-PRL,
polyclonal anti-CypB, or normal rabbit serum as a negative control.
Immunoprecipitated complexes were analyzed by immunoblot analysis with
anti-CypB (1:1000) or anti-PRL (1:1000) and developed as described
above. Where indicated, all quantitation was performed using scanning
densitometry as previously described (5). After densitometric scanning,
ImageQuaNT software (Molecular Dynamics, Inc., Sunnyvale,
CA) was used to quantitate the volume of each band within a given blot
with appropriate background subtraction. All values were normalized to
a CypB or PRL control of known concentration, as appropriate.
For Jak2 and Stat5 activation assays, resting Nb2 cells (PRL-dependent,
rat T cell lymphoma) were incubated with PRL (20 ng/ml), CypB (2
µg/ml), or a combination of the two for 60 min at 37 C. Anti-Jak2 or
-Stat5a (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
immunoprecipitates from the lysate of 4 x
107 cells were analyzed by immunoblot analysis
with the antiphosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY). The blots were
subsequently stripped and reprobed with anti-Jak2 or -Stat5a.
To assess the internalization of the CypB/PRL complex, resting Nb2
cells (3 x 106) were pelleted and cultured
in defined medium containing recombinant human PRL and/or V5
epitope-tagged CypB as above and cultured at 37 C. Cells were harvested
at various intervals, stringently washed, and lysed, as previously
described (9). These lysates were immunoprecipitated with antiserum
against PRL and subjected to immunoblot analysis, as above, with a
horseradish peroxidase (HRP)-conjugated anti-V5 antibody.
To quantify the PRL/CypB interaction in vitro, 50 ng of GST
or GST-CypB expressed in E. coli were coupled to
glutathione-Sepharose beads (Amersham Pharmacia Biotech
followed by a 2-h incubation with 50 ng V5/His-tagged PRL in 1x
binding buffer containing 5 mM
CaCl2 at room temperature. Unbound PRL
(i.e. flow-through) was immunoprecipitated with 1 µg of
polyclonal antihistidine antibody as stated above. PRL bound to the
GST-CypB as well as the immunoprecipitated PRL flow-through were
subjected to immunoblot analysis with anti-V5-HRP (1:1000), and
quantitation was performed by scanning densitometry. The percent of PRL
bound to GST-CypB was determined by comparison with the total amount
recovered. To assess the percentage of PRL bound to CypB in
vivo in human serum, 1 ml of normal donor serum (n = 3) was
immunoprecipitated with a rabbit anti-CypB or anti-PRL polyclonal
antiserum. After washing, the quantity of PRL found in each
immunoprecipitate was determined by a heterogenous sandwich magnetic
separation immunoassay (Bayer Corp., Tarrytown,
NY).
To determine the relative quantities of endogenous CypB present in Nb2
and T47D cells before and after stimulation, rested Nb2 and T47D cells
were stimulated for 2 h at 37 C in the presence of 50 ng/ml PRL
(Nb2) or 50 ng/ml PRL + 10 ng/ml EGF (T47D). After stimulation,
cytoplasmic and nuclear extracts from 7.5 x
105 (Nb2) and 5 x 104
(T47D) rested and stimulated cells were subjected to immunoblot
analysis with anti-CypB (1:1000).
Cell Culture and Cell Proliferation Assays
Nb2 cells (5 x 104 cells per
100-µl well) were plated on 96-well plates in a defined
DMEM-serum free medium (0.1 mM 2-mercaptoethanol, 1%
penicillin/streptomycin, and 1% ITS+; Collaborative Research, Waltham, MA). Cells were rested for 24 h at 37 C
in this defined medium in the absence of PRL before the addition of
5500 pM PRL (either as human pituitary isolated PRL,
recombinant human PRL from E. coli, or recombinant human PRL
from S2 cells; each preparation produced similar results) alone or
premixed with 1- to 2000-fold excess of purified CypB. Parallel studies
were performed using either bGH, recombinant bGH, recombinant rGH, hGH
(all from NIDDK), or IL-2 with Nb2 cells and IL-3 with the murine
IL-3-dependent pre-B lymphoma, Ba/F3. Cultures were incubated for
48 h without refeeding or 72 h with refeeding (100 µl DMEM
with ITS+) at 48 h at 37 C. Proliferation was evaluated by the
addition of 0.5 µCi of 3H-thymidine for 4
h followed by harvesting and scintillation counting while cell
viability was ascertained by trypan blue exclusion. The human breast
cancer line T47D was maintained in DMEM with 10% FBS, while the murine
IL-3-dependent 32D lymphoblastoid line was maintained in RPMI 1640
medium with 10% FBS and 1 ng interleukin-3/ml (PeproTech, Rocky Hill,
NJ). For the PRL and CypB internalization studies, the 32D line
was cultured overnight in RPMI 1640 with ITS+, before use. For
competitive inhibition studies with CypBNT(-), bioassays were performed
as above with 500 pM PRL, 50
nM CypB and 5500 nM
CypBNT(-).
Nuclear Extract Preparation
Nuclear and cytoplasmic extracts were prepared as previously
described (63). Briefly, 3 x 106 rested Nb2
cells in defined medium were stimulated for varying intervals with 50
ng hPRL/ml and 500 ng CypB/ml. After harvesting and washing, the cells
were lysed in 50 µl buffer A (10 mM HEPES, pH 8.0, 1.5
mM MgCl2, 10 mM KCl, 0.5
mM dithiothreitol, 200 mM sucrose, 0.5
mM phenylmethylsulfonyl fluoride (PMSF), 0.5 µg
leupeptin/ml, 0.5 µg aprotonin/ml, 1 mM sodium
orthovanadate, 0.5% NP-40). The lysates were incubated 15 min on ice
and pelleted at 11,000 x g for 10 min and the
cytoplasmic supernatant was transferred to a new tube. The remaining
nuclear pellets were washed twice with buffer A and resuspended in
buffer B [20 mM HEPES, pH 8.0, 20% (vol/vol)
glycerol, 0.1 M KCl, 0.2 mM
EDTA, 0.5 mM dithiothreitol, 0.5
mM PMSF, 0.5 µg leupeptin/ml, 0.5 µg
aprotonin/ml, 1 mM sodium orthovanadate]. These
extracts were subsequently passed three times through a 26 g
needle and centrifuged 30 sec at 14,000 x g. The
pelleted debris was discarded, while the nuclear extract and
cytosolic supernatants were used for immunoblot analysis. Analysis of
the nuclear and cytosolic supernatants revealed <1%
cross-contamination of these fractions (our unpublished
observations).
Indirect Immunofluorescence Microscopy and Confocal Scanning
Microscopy
T47D human breast cancer cells were cultured on glass slides at
approximately 50% confluency in defined DMEM-supplemented ITS+ for
24 h before the addition of 10 ng/ml EGF and combinations of 10
ng/ml PRL ± 100 ng/ml CypB or CypB-NT for 4 h. Nb2 cells
were similarly stimulated in suspension, followed by placement on
slides via cytocentrifugation. After washing, the cells were fixed with
2% paraformaldehyde/0.1% Triton X-100 followed by staining with
anti-PRL diluted in 1% PBS for 1 h at room temperature. The cells
were washed three times with PBS, followed by staining with a
fluorescein isothiocyanate-conjugated goat antirabbit IgG (Roche Molecular Biochemicals, Indianapolis, IN) diluted in 1%
PBS/albumin solution for 1 h at room temperature. Images for
standard immunofluorescence were acquired with IPLab Spectrum software
(Signal Analytics Corp., Vienna, VA). For the delineation of nuclear
DNA for confocal microscopy, cells labeled as above were incubated with
150 U RNase A/ml for 20 min at room temperature, before incubation with
5 µg propidium iodide/ml. Confocal microscopy was conducted at the
Biomedical Imaging Core Laboratory at the University of Pennsylvania
Medical Center, using a TE-300 inverted microscope (Nikon,
Melville, NY) coupled to a 1024-MP confocal imaging system
(Bio-Rad Laboratories, Inc., Richmond, CA). All cells were
optically sectioned in 1-µm steps.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Erica Golemis for her generous assistance with
yeast two-hybrid technology, Dr. David Goodman for his quantitative PRL
immunoassays, and Dr. Leslie Shaw for his gift of CsA. We are also
grateful to Drs. Mark Lemmon, Judy Meinkoth, and Bryan Wolf for their
critical reading of this manuscript and helpful discussions. We thank
the National Hormone and Pituitary Program of the NIDDK for their
generous supply of hormones. We are thankful to Dr. Jim Sanzo for his
assistance in confocal microscopy.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Charles V. Clevenger, M.D., Ph.D., Department of Pathology & Laboratory Medicine, University of Pennsylvania Medical Center, 513 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail:
clevengc{at}mail.med.upenn.edu
Supported by NIH Grant CA-69294 and ACS Grant RPG 00-307-01TBE (to
C.C.). M.A.R. is supported by NIH Training Grant T32 CA-09140 and NRSA
F32 DK 10043-01.
Received for publication July 9, 1999.
Revision received January 14, 2000.
Accepted for publication May 3, 2000.
 |
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