Role of Cyclophilin B in Prolactin Signal Transduction and Nuclear Retrotranslocation

Michael A. Rycyzyn, Sean C. Reilly, Kerri O’Malley and Charles V. Clevenger

Department of Pathology and Laboratory Medicine University of Pennsylvania Medical Center Philadelphia, Pennsylvania 19104


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1Go, the interaction between PRL and CypB in yeast was specific, as determined by several positive and negative controls.


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Table 1. Interaction between PRL and CypB as Assessed by Yeast Two-Hybrid Screening

 
To confirm that CypB directly interacts with PRL, coimmunoprecipitation studies using recombinant CypB and purified PRL were performed on the admixed proteins (Figs. 1AGo and Fig. 3EGo). 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. 3EGo). 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. 1DGo). Further confirmation of the CypB-PRL interaction in vivo was obtained by the direct coimmunoprecipitation of PRL with CypB from human serum (Fig. 1EGo). 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.

 
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. 1BGo). Indeed, this dose-dependent, biphasic effect was most prominent at physiological concentrations (5–10 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. 1CGo). 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. 2Go). 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.

 
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. 3AGo) 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. 3AGo, panel b). The nuclear retrotranslocation of PRL, however, was significantly enhanced by the inclusion of CypB into this defined medium (Fig. 3AGo, panel c), resulting in 95–100% of the T47D nuclei demonstrating anti-PRL immunofluorescence within 2–4 h after addition of PRL and CypB. These data were secondarily confirmed in both T47D (Fig. 3BGo) 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. 3CGo). 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. 3DGo). 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. 3FGo). 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. 4AGo). 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. 4BGo). 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. 3Go, A and B) with a dose-dependent loss of enhanced PRL-driven 3H-thymidine incorporation (Fig. 4CGo). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 2–12 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 manufacturer’s 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 5–500 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 5–500 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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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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