Prolactin Stimulates Activation of c-jun N-Terminal Kinase (JNK)

Kathryn L. Schwertfeger, Seija Hunter, Lynn E. Heasley, Valerie Levresse, Ronald P. Leon, James DeGregori and Steven M. Anderson

Departments of Pathology (K.L.S., S.H., S.M.A.), Medicine (L.E.H., V.L.), Biochemistry (R.P.L., J.D.), Pediatrics (J.D.), and Program in Molecular Biology (K.L.S., J.D., S.M.A.) University of Colorado Health Sciences Center Denver, Colorado 80262


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In recent years the mitogen-activated protein (MAP) kinase family has expanded to include both c-jun N-terminal kinases (JNKs), and the p38/HOG1 family in addition to the extracellular regulated kinase (ERK) family. These kinases are activated by a variety of growth factors, as well as extra- and intracellular insults such as osmotic stress, UV light, and chemotherapeutic agents. Stimulation of the PRL-dependent Nb2 cell line with PRL results in the rapid activation of JNK as determined by the glutathione-S-transferase (GST)-jun kinase assay. Activation was maximal 30 min after stimulation with 50 nM rat PRL (rPRL) and decreased after that time. Dose response studies indicated that concentrations as low as 10 nM rPRL resulted in maximal activation. The interleukin-3 (IL-3)-dependent myeloid progenitor cell line 32Dcl3 was transfected with the long, Nb2, and short forms of the rat PRL receptor (rPRLR), as well as the long form of the human PRLR (hPRLR). The long and Nb2 forms of the PRLR were able to stimulate activation of JNK; however, the short form of the rPRLR was not. This corresponds with the inability of the short form of the rPRLR to stimulate proliferation of 32Dcl3 cells. Activation of JNK in 32Dcl3 cells expressing the long form of the hPRLR was maximal at 30 min after stimulation with 100 nM ovine PRL (oPRL) and declined after that time. Dose response studies indicated that activation of JNK was maximal after 30 min at a concentration of 10 nM, and the amount of activated JNK declined at the highest concentration of oPRL, 100 nM. Immunoblot analysis with an antibody that recognizes the activated (phosphorylated) forms of JNK1 and JNK2 indicated that both JNK1 and JNK2 isoforms were activated in 32D/hPRLR cells stimulated with oPRL. A recombinant human adenovirus expressing a kinase-inactive mutant of JNK1 (APF mutant) was used to determine the biological effect of blocking JNK activity in Nb2 cells. Expression of the JNK1-APF mutant inhibited cellular proliferation and induced DNA fragmentation typical of cells undergoing apoptosis. These data suggest that activation of JNKs may be important in mitogenic signaling and/or suppression of apoptosis in Nb2 cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mitogen-activated protein (MAP) kinases were initially discovered as low mol wt protein kinases that were rapidly activated after stimulation of cells with a variety of mitogens. In recent years this family of serine/threonine-specific protein kinases has expanded dramatically and now includes protein kinases that are activated by a variety of cellular stresses in addition to mitogenic stimulation. The original members of this family are alternatively known as MAP kinase-1and MAP kinase-2, or extracellular related kinase 1 and 2 (ERK-1 and ERK-2) (1). These two kinases are the prototypes for all MAP kinases, and they share structural features with two more recently discovered families: the JNK/SAPK (c-jun N-terminal kinase/stress-activated protein kinase) family (2, 3, 4, 5, 6, 7), and the p38 family (8, 9). All MAP kinases are proline-directed serine/threonine protein kinases that are activated by the phosphorylation of both a threonine and a tyrosine residue in a Thr-X-Tyr motif in the activation loop that lies in close proximity to the ATP- and substrate-binding sites. Although MAP kinases are thought to have a similar structure, they differ in the size of the activation loop, and the amino acid present in the second position of the Thr-X-Tyr motif; ERKs have a Glu, JNKs have a Pro, and p38 family members have a Gly in the second "X" position.

ERK-1 and ERK-2 have been demonstrated to be critical in mitogenic signaling and oncogenesis (10, 11). After the phosphorylation of these two kinases, some of the ERK-1 and ERK-2 translocate to the nucleus (12), where they have been shown to phosphorylate transcription factors such as ternary complex factor (13). These transcription factors are required for the transcription of the immediate early response gene c-fos (14). Thus, MAP kinases tie signals initiated at the plasma membrane by growth factor receptors to the induction of immediate early response genes in the nucleus, which are required for progression through the cell cycle. Activation of ERK-1 and ERK-2 is also important in stimulating differentiation of some cell types. Although it is not currently understood what distinguishes a proliferative signal from one that induces differentiation, some investigators have suggested that the length of time MAP kinase remains activated is important (15). There is also evidence that activation of ERKs stimulates protection against factors that mediate apoptosis (16, 17).

The JNK family is defined based upon the ability of these kinases to phosphorylate serine residues 63 and 73 in the N-terminal region of c-jun (18), an immediate early gene product that is a component of the AP1 transcription factor complex (19). c-jun is preferentially phosphorylated over either junB or junD, because c-jun contains a docking site for JNKs (19). JNKs have also been described as SAPKs since they were activated by a variety of cellular stresses including UV light, heat, hyperosmotic shock, reactive oxygen species, antioxidants, protein synthesis inhibitors, and inflammatory cytokines (interleukin-1 and tumor necrosis factor) (2, 8, 20, 21, 22, 23, 24). There are currently three members of the JNK/SAPK family: JNK-1, JNK-2, and JNK-3 which correspond to SAPK-{gamma}, -{alpha}, and -ß, respectively (2, 4). Some investigators have argued that JNK1 and JNK2, but not ERK-1 and ERK-2, are required for induction of c-fos and c-jun (24); however, other studies support a role for ERKs in this process. JNKs are activated by stimulation of cells with mitogenic cytokines, although it is not clear whether their activation is required for mitogenesis (25, 26, 27).

The PRLR is a member of the cytokine receptor superfamily. The ability of several cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and erythropoietin, to activate JNKs stimulated us to ask whether JNKs represented another class of signaling molecules activated by the PRLR. In this study we describe the ability of PRL to induce activation of JNKs. A time- and dose-dependent activation of JNK was observed in the PRL-responsive T cell line Nb2, and in 32Dcl3 cells transfected with the long and Nb2 forms of the rPRLR. Expression of a kinase-inactive mutant of JNK1 in Nb2 cells inhibited cellular proliferation and induced apoptosis as indicated by the induction of DNA fragmentation typical of apoptotic cells. These data suggest that JNKs represent another signaling family activated in response to PRL stimulation that may contribute to mitogenic signaling and/or the suppression of apoptosis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL Stimulates Activation of JNK
PRL has been reported to activate ERK1 and ERK2, two members of the MAP kinase family (28, 29, 30). Numerous studies have indicated that mitogenic growth factors activate another member of the MAP kinase family, the c-jun N-terminal kinases or JNKs, in addition to ERK1 and ERK2; these studies have included mitogenic cytokines that stimulate proliferation of hematopoietic cells (25, 26, 27). These observations prompted us to determine whether PRL might also stimulate activation of JNK family members. This question was first examined in Nb2 cells, a PRL-dependent rat T cell line. Stimulation of Nb2 cells with 50 nM rPRL for 0–60 min resulted in the rapid activation of JNK as determined by the glutathione S-transferase (GST)-jun kinase assay (Fig. 1AGo). Although a basal level of JNK activity was detectable in unstimulated Nb2 cells, PRL stimulation resulted in an increase in JNK activity within 5 min. JNK activity was maximal 30 min after stimulation of Nb2 cells and declined after that until 60 min when it was at or below the level detected in unstimulated cells (Fig. 1AGo). A dose response curve indicated that concentrations of as low as 5 nM rPRL resulted in JNK activation, and that higher concentrations of PRL did not substantially increase JNK activation after 15 min as measured by the GST-jun kinase assay (Fig. 1BGo). These results clearly indicate that PRL can activate JNK.



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Figure 1. PRL Induces Activation of JNK in Nb2 Cells

A, Nb2 cells were cultured overnight in medium supplemented with 10% horse serum and were then concentrated and stimulated with 50 nM rPRL for 0 to 60 min. The cells were then lysed and processed as described to examine the activation of JNK as determined by the GST-jun kinase assay. The time in minutes is shown at the top of the panel. B, Nb2 cells were stimulated for 15 min with 0–100 nM rPRL, after which the cells were processed as described above. The concentration of rPRL used to stimulate the cells is shown at the top of each lane. The arrowhead on the right side of each panel indicates the position of the GST-jun fusion protein.

 
To demonstrate the physiological relevance of PRL-induced JNK activation in a cell type that is responsive to PRL, we examined the activation of PRL in the human breast cancer cell line T47D. Stimulation of T47D cells with PRL has been shown to result in the activation of the JAK2 tyrosine kinase and the phosphorylation of STAT molecules (31). Stimulation of T47D cells with 50 nM ovine PRL (oPRL) resulted in the activation of JNK as determined by the GST-jun kinase assay (Fig. 2Go, lanes 1–6). Maximal levels of JNK activity were evident 30 min post stimulation and declined after that time. A dose response study indicated that 5 nM oPRL resulted in maximal activation of JNK and higher concentrations of oPRL did not stimulate either an increase or decrease in JNK activity (Fig. 2Go, lanes 7–12). Immunoblotting with phospho-JNK antibodies indicated that both JNK1 and JNK2 were activated (data not shown).



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Figure 2. PRL Induces Activation of JNK in T47D Cells

T47D cells were cultured overnight in serum-free medium and then stimulated with 50 nM oPRL for 0–60 min (lanes 1–6). The cells were then lysed and processed as described to examine the activation of JNK as determined by the GST-jun kinase assay. The time in minutes is shown at the top of each lane. In lanes 7–12, T47D cells were treated in an identical manner and then stimulated for 30 min with 0–100 nM oPRL, and processed as described above. The concentration of rPRL used to stimulate the cells is shown at the top of each lane. Lane number is indicated at the bottom of each lane. The arrowhead on the right side of the panel indicates the position of the GST-jun fusion protein.

 
Only the Long and Nb2 Forms of the rPRLR Are Able to Activate JNK
The PRLR is expressed in multiple forms that result from differential splicing (32, 33, 34, 35). A unique form of the PRLR has been identified in the Nb2 cell line; this form is transcribed from a PRLR gene unique to this cell line that has undergone an internal deletion (36). O’Neal and Yu-Lee (37) have reported that both the long and Nb2 forms of the rPRLR are able to induce PRL-dependent proliferation when introduced into the IL-3-dependent murine myeloid cell line FDCP-1. In contrast, the short form of the rPRLR was not able to induce proliferation in their system (37). Likewise we have observed that the long and Nb2 forms of the rPRLR, but not the short form, are able to induce PRL-dependent proliferation when introduced into 32Dcl3 cells (data not shown). We were therefore interested in determining whether each of these three forms of the rPRLR were able to induce JNK activation. We generated a panel of 32Dcl3 cells expressing a single form of the rPRLR which was tagged with the FLAG epitope tag such that we could demonstrate expression of the different forms of the PRLR. Consistent with the results of previous investigators (38, 39), the short, Nb2, and long forms of the rPRLR were all able to induce activation of JAK2 as assessed by immunoprecipitation of JAK2 from stimulated cells followed by immunoblotting with antiphosphotyrosine (data not shown). Phosphorylation of tyrosine1007 in the activation loop of JAK2 is required for the catalytic activation of JAK2 (40) and is detected by antiphosphotyrosine immunoblotting. The different transfected cell lines, as well as untransfected control cells, were cultured overnight in medium supplemented with 2% charcoal-stripped serum to allow signaling molecules to return to their basal level. Only the long and Nb2 forms of the PRLR stimulated activation of JNK as determined by the GST-jun kinase assay (Fig. 3AGo). No PRL-induced activation of JNK was observed in the untransfected 32Dcl3 cells or in the 32D/short cells (Fig. 3AGo). Expression of the FLAG-tagged versions of the rPRLR was detected in the different transfected cells (Fig. 3Go, B–D). We cannot comment on the relative amounts of the different receptors in the clones of the transfected cells since different approaches were used to detect expression of the protein molecules in the different clones, although other clones prepared in a similar manner in our laboratory express 3,000–5,000 receptors per cell. O’Neal and Yu-Lee (37) observed that the Nb2 form of the rPRLR stimulated a more robust proliferation of FDCP-1 cells than the long form of the rPRLR. We have performed dose-response studies that indicate that our 32D/Nb2 cells respond to lower concentrations of PRL and exhibit a greater extent of proliferation as measured by an MTT [3-(4, 5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide] assay when compared with 32D/long cells (data not shown). However, as noted above, we cannot directly compare the amount of protein for the long and Nb2 forms of the PRLR in the transfected cells; therefore, we cannot eliminate the possibility that there is simply more Nb2 protein in cells transfected with that form of the PRLR.



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Figure 3. Activation of JNK in 32Dcl3 Cells Expressing Different Forms of the rPRLR

A, The 32Dcl3 cell line was transfected with the long, Nb2, or the short form of the rPRLR, and single cell-derived clones were isolated. Parental 32Dcl3 cells (lanes 1–4), 32D/long (lanes 5–8), 32D/Nb2 (lanes 9–12), and 32D/short cells were stimulated with 10 nM rPRL for 0–60 min, the cells were lysed, and the GST-jun kinase assay was used to examine the activation of JNKs. The time of stimulation in minutes is indicated at the top of each lane. The arrowhead on the right side of the panel indicates the position of the GST-jun fusion protein. B, 32Dcl3 and 32D/long cells were lysed and membrane fractions were prepared. Total protein (100 µg) was resolved on a 7.5% polyacrylamide gel. The immunoblot was probed with anti-Flag M2 antibody. C, 32Dcl3 and 32D/Nb2 cells were lysed and 100 µg total cellular protein were resolved on a 7.5% polyacrylamide gel. The immunoblot was probed with the anti-Flag M2 antibody. D, 32Dcl3 and 32D/short cells were lysed, immunoprecipitated with the anti-Flag M2 antibody, and resolved on a 10% polyacrylamide gel. The immunoblot was probed with anti-M2 antibody.

 
The long form of the human PRLR was also transfected into 32Dcl3 cells. Stimulation of the 32D/hPRLR cells with 100 nM oPRL resulted in the rapid activation of JNK (Fig. 4AGo). Although JNK activation could be detected after 5 min, it was more apparent at 15 min post stimulation. Activation was maximal at 30 min post stimulation and declined afterward until reaching basal levels at 1 h (Fig. 4AGo, lanes 1–6). A dose response study with 32D/hPRLR cells yielded slightly different results than obtained in Fig. 1Go. JNK activation could be detected with 5 nM oPRL, and the maximal activation was observed at 10 nM oPRL (Fig. 4AGo, lanes 7–12). Concentrations higher than 10 nM actually were observed to result in lower levels of JNK activation than detected with 10 nM (Fig. 4AGo). This result is consistent with dose-response curves for PRL-induced mitogenesis as described previously, and results from the fact that PRL binds to the PRLR as a monomer through two different binding sites and at high concentrations of PRL, receptor dimerization is actually inhibited (41). Clearly the bell-shaped dose-response curve can be influenced by many factors including ligand affinity for both sites on the receptor, the type and species of the cell studied, the exact response being investigated, the number of receptors upon the cell, and the sensitivity of the bioassay used. As a basis of comparison, the 32D/hPRLR cells were also stimulated with 100 U/ml IL-3, a concentration 5-fold greater than that needed to stimulate maximal proliferation of 32Dcl3 cells, and activation of JNK was examined. Consistent with previous results by other investigators (25, 26), IL-3 stimulated the activation of JNK as determined by the GST-jun kinase assay (Fig. 4BGo). In contrast to the results obtained with PRL-stimulated activation of JNK in Figs. 1Go, 3Go, and 4AGo, IL-3 appeared to stimulate JNK activation for a longer period of time (Fig. 4BGo); however, the physiological significance of this is not clear.



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Figure 4. PRL Induces Activation of JNK in 32Dcl3 Cells Transfected with the Human PRL Receptor

A, 32D/hPRLR cells were cultured overnight in medium supplemented with 2% charcoal-stripped serum and then concentrated and stimulated with 100 nM oPRL for 0–60 min (lanes 1–6) or stimulated with 0–100 nM oPRL for 10 min (lanes 7–12). The cells were then lysed and processed as described to examine the activation of JNK as determined by the GST-jun kinase assay. The time in minutes is shown at the top of lanes 1–6, and the concentration of oPRL is indicated at the top of lanes 7–12. Lane numbers are indicated at the bottom of the panel. B, 32D/hPRLR cells were stimulated for 0–60 min with 100 U/ml recombinant murine IL-3, and the cells were then processed as described above. The time of stimulation in minutes is indicated at the top of each lane. The arrowhead on the right side of each panel indicates the position of the GST-jun fusion protein.

 
Activation of JNKs and ERKs Occurs with Similar Kinetics
Activation of ERKs has been described as being critical in signaling pathways that regulate cellular proliferation. We therefore were interested in comparing the kinetics of ERK and JNK activation in 32Dcl3 cells expressing the long form of the hPRLR. These cells were stimulated with either 10 nM oPRL or 100 U/ml IL-3 for 0–60 min, and activation of both JNKs and ERKs was examined by immunoblotting with antibodies that specifically recognize the activated forms of these enzymes (Fig. 5Go). Activation of JNKs was maximal at 15–30 min post stimulation with either oPRL or IL-3 (Fig. 5AGo). Some activated JNK was observed in unstimulated cell control for the IL-3 study (Fig. 5AGo, lane 7) which was surprising since they were treated in a manner identical to the control cell for PRL stimulation (Fig. 5AGo, lane 1). We do not routinely detect a basal level of activated JNK in 32Dcl3 cells. Bands corresponding to both p46 JNK1 and p54 JNK2 were observed in cells stimulated with either oPRL or IL-3. Minor bands that migrated below and above the Mr 46,000 band were observed after 15 min stimulation with either oPRL or IL-3. These bands may correspond to minor splice variants of different JNK family members. Reprobing the immunoblot with an antibody that recognizes both JNK1 and JNK2, although primarily the latter, revealed the presence of equal amounts of JNK1 and JNK2 in all samples examined. Similar studies have been conducted with an antibody that recognizes primarily JNK1, demonstrating no changes in the amount of JNK1 protein over the time period investigated in this study (data not shown).



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Figure 5. Comparison of the Activation of Both JNK and ERK by Either PRL or IL-3

32Dcl3 cells expressing the long form of the human PRLR were stimulated with either 10 nM oPRL (lanes 1–6), or 100 U/ml IL-3 (lanes 7–12) for 0–60 min. Cell lysates were prepared and 50 µg total cellular protein were resolved on an 10% polyacrylamide gel. The immunoblot was probed with anti-phospho-JNK, which recognizes activated JNK1 and JNK2 (panel A); anti-JNK2, which cross-reacts with JNK1 (panel B); anti-ACTIVE ERK2, which cross-reacts with ERK1 (panel C); and anti-ERK which detects both ERK1 and ERK2 (panel D). The time in minutes is shown at the top of each lane. The arrowheads on the right side of the panel indicate the positions of JNK-1, JNK-2, ERK-1, and ERK-2.

 
The same approach was used to examine the activation of both ERK1 and ERK2 after stimulation with either oPRL or IL-3. PRL stimulation resulted in the activation of both ERK1 and ERK2 as early as 5 min after stimulation (Fig. 5CGo, lanes 1–6). PRL stimulated maximal activation of ERK1 and ERK2 after 15 min, after which the amount of activated ERK1 and ERK2 declined (Fig. 5CGo, lanes 1–6). Stimulation with IL-3 resulted in a similar pattern of ERK activation with maximal activation evident at 15 min post stimulation (Fig. 5CGo, lanes 7–12). Equal amounts of ERK1 and ERK2 were demonstrated by probing the immunoblot with an antibody that recognizes both ERK1 and ERK2 (Fig. 5DGo). Based upon this method of analysis, there does not appear to be any major difference in the activation of JNKs and ERKs by either PRL or IL-3.

A Kinase-Inactive Mutant of JNK1 Inhibits PRL-Induced Proliferation
The data presented above indicate that PRL induces activation of JNK1 and JNK2; however, these studies do not provide any indication of the biological importance of JNK activation. We have chosen to use a kinase-inactive mutant of JNK1 to address this point. As noted above, activation of JNK family members requires the phosphorylation of both tyrosine and threonine residues in the activation loop of these kinases (42). Mutation of these two residues to nonphosphorylatable amino acids results in the generation of nonactivatable kinases that are able to suppress the activation of the endogenous JNKs (20, 21). Such dominant negative mutants have been used to address the biological importance of JNKs in biological processes: Butterfield et al. (20) used the JNK1-APF and JNK2-APF mutants to demonstrate the importance of JNKs in regulation of UV-induced apoptosis in small cell lung cancer cell lines. The JNK1-APF mutant cDNA was cloned into a recombinant human adenovirus by overlap recombination (43), to generate a virus capable of transducing the dominant negative JNK1 mutant into a variety of cells. Nb2 cells, like other T lymphocyte cells, cannot be infected with human adenoviruses due to the absence of the viral receptor protein coxsackie-adenovirus receptor (CAR) (44). Therefore, a recently described deletion mutant of CAR, CAR{Delta}1 (44), was introduced into Nb2 cells to allow transduction of these cells with recombinant human adenoviruses. Transduction of Nb2/CAR{Delta}1 cells with a recombinant human adenovirus expressing green fluorescent protein (Adeno-GFP) results in >95% of the cells expressing GFP (data not shown).

The effect of the JNK1-APF mutant upon the proliferation of Nb2 cells was examined by transducing Nb2/CAR{Delta}1 and control Nb2 cells with varying amounts of the virus ranging from a multiplicity of infection (MOI) of 0–5. As shown in Fig. 6Go, transduction of Nb2/CAR{Delta}1 cells with Adeno-JNK1-APF virus caused a dramatic decrease in the number of viable cells over a 3-day period. There were consistently fewer cells in cultures of Nb2/CAR{Delta}1 cells transduced with the JNK1-APF virus at all time points examined at both MOIs of 2.5 or 5. This decrease occurred in a dose-dependent manner with fewer cells being present in cultures transduced at a MOI of 5 compared with those transduced at a MOI of 2.5. There was no significant difference in the number of Nb2/CAR{Delta}1 cells transduced with Adeno-GFP compared with normal control cells. These studies were done on cells cultured in the presence of 200 ng/ml (10 nM) PRL; however, identical results were obtained when the cells were cultured in complete media containing FCS (data not shown).



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Figure 6. Kinase-Inactive JNK1-APF Mutant Blocks PRL-Induced Proliferation of Nb2/CAR{Delta}1 Cells

Nb2/CAR{Delta}1 cells were either left untransduced ({diamondsuit}) or transduced with the recombinant human adenovirus encoding the JNK1-APF mutant at a MOI of 2.5 ({blacksquare}), the JNK1-APF mutant at a MOI of 5 ({blacktriangleup}), the Adeno-GFP at MOI of 2.5 (X), or Adeno-GFP at a MOI of 5 () for 24 h. The cells were then counted and cultures initiated at a concentration of 2 x 105 cells per ml in medium containing 200 ng/ml oPRL. At 24-h intervals the number of viable cells was determined by trypan blue exclusion.

 
To demonstrate the effect of the JNK1-APF mutant on the activation of the endogenous JNK1, Nb2 and Nb2/CAR{Delta}1 cells were transduced at MOIs of 0, 0.5, and 5 with either the Adeno-JNK1-APF or the Adeno-GFP. Transduction of Nb2/CAR{Delta}1 cells with the JNK1-APF mutant at a MOI of 5 suppressed the level of activated JNK1 after PRL stimulation as determined by immunoblotting with anti-phospho-JNK antibody (Fig. 7AGo, lane 9). There did not appear to be a decrease in the activation of JNK1 in cells transduced with the JNK1-APF virus at a MOI of 0.5 compared with mock infected cells (Fig. 7AGo, lanes 7 and 8), nor did transduction of Nb2/CAR{Delta}1 cells with Adeno-GFP suppress PRL-induced JNK1 activation (Fig. 7AGo, lanes 10–12). Likewise, transduction of Nb2 cells with either the Adeno-JNK1-APF or Adeno-GFP viruses had no effect upon the activation of JNK1 (Fig. 7AGo, lanes 1–6). Expression of the hemagglutinin (HA)-tagged JNK1 protein was only detected in cells transduced with the JNK1-APF mutant at a MOI of 5 (Fig. 7BGo, lane 9). An endogenous background band is seen in all lanes migrating just above the HA-tagged JNK1-APF protein. This band is seen in all cells, including the mock-transduced control cells (Fig. 7BGo, lanes 1, 4, 7, and 10). We interpret these data to indicate that transduction of Nb2/CAR{Delta}1 cells with the Adeno-JNK1-APF virus suppresses the PRL-induced activation of JNK1 and suppresses the PRL-induced proliferation of Nb2/CAR{Delta}1 cells.



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Figure 7. Kinase Inactive JNK1-APF Mutant Blocks PRL-Induced JNK Activation in Nb2/CAR{Delta}1, but Not Nb2 Cells

Nb2 (lanes 1–6) and Nb2/CAR{Delta}1 (lanes 7–12) were transduced with the recombinant human adenovirus encoding the JNK1-APF mutant (lanes 1–3 and 7–9) or Adeno-GFP (lanes 4–6 and 10–12), at a MOI of 0 (lanes 1, 4, 7, and 10), MOI of 0.5 (lanes 2, 5, 8, and 11), or a MOI of 5 (lanes 3, 6, 9, and 12) for 24 h in Fisher’s medium supplemented with 10% horse serum and 200 ng/ml oPRL. The cells were lysed and clarified, and the protein concentration was determined as described above. A 50-µg sample of each lysate was resolved on a 10% SDS polyacrylamide gel, and the proteins were transferred to a nylon membrane and then immunoblotted with anti-phospho-JNK1 (panel A), or anti-HA monoclonal antibody (panel B). The positions of phospho-JNK1 and the HA-tagged JNK1-APF proteins are indicated on the right side of the panel. The virus and MOI are indicated at the top of each lane.

 
Kinase-Inactive Mutant of JNK1 Induces Apoptosis of Nb2 Cells
The observed decrease in cellular proliferation described in Fig. 6Go above could result from a decrease in mitogenic signaling or an increase in apoptosis. To determine whether there was an increase in apoptotic cells, we looked for whether there was an increase in DNA fragmentation typical of cells undergoing apoptosis. Nb2 and Nb2/CAR{Delta}1 cells were transduced with the Adeno-JNK1-APF virus at MOIs varying from 0–5. After 24 h the cells were harvested from the cultures, the DNA isolated, and the fragmentation of the DNA assessed by resolving a 10-µg sample of each DNA on a 1.5% agarose gel. As can be seen in Fig. 8Go, transduction of the Nb2/CAR{Delta}1 cells, but not the Nb2 cells, resulted in the induction of a DNA ladder typical of cells undergoing apoptosis. Transduction of Nb2/CAR{Delta}1 cells with recombinant human adenovirus encoding GFP at the same MOI did not induce DNA fragmentation.



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Figure 8. Kinase-Inactive JNK1-APF Mutant Induces DNA Fragmentation in Nb2/CAR{Delta}1, but Not Nb2 Cells, Grown in the Presence of PRL

Nb2/CAR{Delta}1 cells were left untransduced (lanes 2 and 5), transduced with the recombinant human adenovirus encoding the JNK1-APF mutant (lanes 3 and 4), or transduced with Adeno-GFP (lanes 6 and 7). A MOI of 2.5 was used in lanes 3 and 6, and a MOI of 5 was used in lanes 4 and 7. After transduction the cells were cultured for 24 h in the presence of 200 ng/ml oPRL before being lysed as described. After purification and quantitation of the amount of DNA in each sample, a 10 µg amount of DNA was resolved on a 1.5% agarose gel. Lane 1 contains size markers.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In recent years the MAP kinase family has grown to include three subfamilies: the ERK family, the JNK family, and most recently the p38/HOG1 family. Current investigations are focused upon determining the role of each of these different kinases in mitogenesis, response to cellular stress, apoptosis, differentiation, and oncogenic transformation. The activation of JNK family members was first demonstrated to occur in response to stresses such as TNF-{alpha} (4), heat shock (4), protein synthesis inhibitors (4, 24), UV light (2, 7, 21), {gamma}-irradiation (21, 45), nitric oxide and hydrogen peroxide (23), and osmotic stress (2). A variety of proliferative signals have also been demonstrated to induce JNK activation, such as the BCR-ABL oncogene (46), and several hematopoietic growth factors, including stem cell factor, IL-3, granulocyte macrophage-colony stimulating factor, erythropoietin, and thrombopoietin (25, 26, 27).

In this study we have demonstrated that PRL induces activation of JNK in a time- and dose-dependent manner. This was observed in a number of different cell lines including the PRL-dependent Nb2 cell line, and an IL-3-dependent murine myeloid cell line stably expressing different forms of the PRLR, and the T47D cell line. Our data suggest that only the long and Nb2 forms of the rPRLR are able to stimulate activation of JNKs, as well as induce proliferation of transfected 32Dcl3 cells. Although other investigators have reported that the short form of the PRLR can induce proliferation of NIH3T3 cells (47), we have not observed any biological effect of this form of the PRLR.

A role for JNK activation in mitogenesis is suggested by the fact that the two forms of the rPRLR that are able to stimulate proliferation (37) are both able to induce activation of JNK. The effect of expression of the JNK1-APF mutant in Nb2 cells also suggests that JNK1 may provide an important mitogenic signal downstream of the PRLR. The fact that the JNK1-APF mutant appears to induce apoptosis suggests that either apoptosis is the default pathway in these cells and in the absence of a mitogenic signal the cells undergo apoptosis, or that JNK1 is important in suppressing apoptosis. Our current studies do not distinguish between these two possibilities. Clearly, JNK inhibition results in a dominant phenotype resulting in cellular death. It would be interesting to determine whether a constitutively activated form of JNK1 would suppress apoptosis or induce growth factor-independent proliferation.

The JNK family members are encoded by three different genes (JNK1, JNK2, and JNK3), and the transcripts of each gene are able to undergo alternative splicing, resulting in the production of multiple protein products (48). Our immunoblotting studies indicate that both JNK1 and JNK2 are activated by PRL, and that there are additional minor splice variants that can be detected by immunoblotting. We have not detected the activation of JNK3 in these cells, which may not be surprising since current evidence suggests that expression of JNK3 may be highest in neuronal tissues (48, 49).

The activation of ERK1 and ERK2 can either stimulate cellular proliferation or differentiation. Although it is not known what biochemical events distinguish a proliferative signal generated by ERKs from one that induces differentiation, it has been suggested that the duration of ERK activity may be involved. Transient activation of ERKs has been suggested to mediate a proliferative signal (15). Depending upon the stimulus, JNK activation may be involved in either mitogenesis, oncogenic transformation, differentiation, or induction of apoptosis. It has been suggested that the transient induction of JNK activity by activation of the T cell receptor may lead to cell proliferation, whereas prolonged induction by certain apoptotic signals leads to apoptosis (21). PRL induces a transient activation of JNK, peaking at 15–30 min and then decreases in all the cell types we examined. Our data indicate that both JNK and ERK are activated with approximately the same rate in several different cell types. It is not clear at this time whether the activation of both JNKs and ERKs is required for PRL-induced mitogenesis or whether each kinase has a unique function. Our data suggest that JNKs represent another class of signaling molecules that lie downstream of the PRLR that may be required for mitogenesis by this receptor. Understanding the role that JNKs play in signaling, and the identification of mechanisms that regulate activation of JNK in response to PRL, is fundamental to our understanding of signaling events that regulate cellular proliferation, differentiation, and cell death.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
The Nb2–11 cell line was obtained from Dr. Arthur Buckley (University of Cincinnati School of Medicine, Cincinnati, OH). The cells were maintained in Fishers medium supplemented with 10% FCS, 10% horse serum, 1 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10-4 M ß-mercaptoethanol. The 32Dcl3 cell line was obtained from Dr. Joel Greenberger (University of Pittsburgh, Pittsburgh, PA). Characterization of 32Dcl3 cells with the human and Nb2 forms of the PRLR were described previously (50). Cultivation of 32Dcl3 cells has been described recently (51).

Charcoal-stripped FCS was obtained from HyClone Laboratories, Inc. (Logan, UT), and FCS was from Summit Biotechnology (Fort Collins, CO). All other media components were from Life Technologies, Inc./BRL (Gaithersburg, MD). Human (lot AFP-3855A), rat (lot APF-6452B), and ovine (lot AFP-10677C) PRL were obtained from the National Hormone and Pituitary Program (Rockville, MD). Recombinant murine IL-3 was obtained from Collaborative Biomedical Products (Bedford, MA).

Kinase Assay for JNK Activity
Cells to be analyzed were cultured overnight in media supplemented with charcoal-stripped serum. Cells (2 x 107) were resuspended in 1 ml serum-free tissue culture medium supplemented with the indicated concentration of either PRL or IL-3 for the indicated times. The cells were pelleted in a microfuge and lysed in JNK lysis buffer [25 mM HEPES, pH 7.7, 20 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1% Triton X-100, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 4 µg/ml Aprotinin (Calbiochem, La Jolla, CA)] and allowed to sit on ice for 30 min. The lysates were clarified by spinning at 10,000 rpm for 5 min in a refrigerated Savant SRF13K microfuge. Protein concentrations of the clarified lysate were determined by the Pierce Chemical Co. Coomassie Plus protein assay (Rockford, IL), and 300 µg total cellular protein were used in each assay. A 100 µl volume of a 10% suspension of GST-c-jun (1–79) was added to 300 µg total cellular protein in a final volume of 1 ml and incubated for 2 h at 4 C. The beads were then washed three times with 20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100. The washed beads were then incubated for 20 min at 30 C, in 40 µl 50 mM ß-glycerophosphate, pH 7.6, 0.1 mM sodium orthovanadate, 10 mM MgCl2, 20 µM ATP (5,000 cpm/pmol). The reactions were terminated by the addition of 10 µl 5 x SDS sample buffer and boiled, and the reaction products were resolved on a 10% SDS polyacrylamide gel. The position of GST-jun was determined by staining the gel, and the extent of GST-jun phosphorylation was determined by autoradiography.

The GST-c-jun (1–79) expression vector and purification of the fusion proteins have been described previously (20). A 5 ml overnight culture of the bacterial was diluted to 500 ml and allowed to grow until an OD600 of 0.6. IPTG was added to a final concentration of 0.4 mM for 3 h, after which the bacteria were collected by centrifugation and the pellets frozen at -20 C. The pellets were thawed, resuspended in 10 ml NETN [20 mM Tris, pH 7.6, 100 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol, 0.5% NP-40, 100 U/ml Aprotinin (Calbiochem)], and 200 µl of 1 mg/ml lysozyme were added. After 1 h on ice, the bacteria were sonicated for 2 min, the lysates clarified by centrifugation at 10,000 rpm for 10 min, and the supernatant fluid transferred to a new tube. A 50% slurry of glutathione agarose (1.5 ml) (Pharmacia Biotech, Piscataway, NJ) was added, and the tube was placed on a rocking platform for 1 h at 4 C. The beads were washed three times with NETN buffer and resuspended in JNK lysis buffer to a concentration of 10%.

Immunoblotting
Cells were lysed in JNK lysis buffer and the lysates clarified by spinning at 10,000 rpm in a Savant RCF13K refrigerated microfuge for 30 min. Protein concentrations were determined as described above. Each cell lysate (50–75 µg) was resolved on a 10% gel, transferred to an Immobilon membrane, and immunoblotted with the desired antibody as described previously (51, 52). Immunoprecipitation with the anti-Flag antibody and preparation of membrane fractions were performed as described previously (53).

Anti-JNK1 and anti-JNK2 and an anti-MAP kinase antibody that cross-reacts with both ERK1 and ERK2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ACTIVE ERK2 antibody was obtained from Promega Corp. (Madison, WI). Anti-phospho-JNK antibody and control anti-JNK antibody were obtained from New England Biolabs, Inc. (Beverly, MA). The anti-HA monoclonal antibody was obtained from Roche Molecular Biochemicals (Indianapolis, IN). The anti-Flag monoclonal antibody M2 was obtained from Sigma (St. Louis, MO).

Generation of Recombinant Human Adenovirus Encoding Kinase Inactivate Mutants of JNK1 and Infection of Nb2 Cells
Recombinant human adenovirus encoding the JNK1-APF mutant was generated by overlap recombination (43). A fragment encoding the JNK1-APF isoform cDNA was ligated between the HindIII and XbaI sites in the pACCMV#95, which encodes the left end of the adenovirus chromosome with the E1A gene and the 5'-half of the E1B gene replaced by the cytomegalovirus major immediate early promoter, a multiple cloning site, intron, and polyadenylation sequences from SV40 to yield the plasmid pACCMV#95-JNK1-APF. The plasmid (5 µg) was mixed with BstBI-digested Ad5dl327Bstß-Gal-TP complex. The mixture was precipitated with calcium phosphate for 1 min in a total volume of 400 µl and then overlaid on 293 cells that were at 50% confluency. After 8–16 h, fresh DMEM supplemented with 10% FCS was added, and the transfected cells were incubated for 7 days. The transfected cells were lysed by three cycles of freezing and thawing, and dilutions of the freeze-thaw lysates were used to infect fresh 293 cells to plaque purify the virus. The infected 293 cells were overlaid with DMEM containing 10% FCS and 1% Noble agar, fed with the same medium 4 days later, and then stained with 5-bromo-4-chloro-3-indoylb-D-galactopyranoside and neutral red. Clear plaques, which are derived from recombinant adenovirus that have lost the LacZ gene present in the parental viral chromosome, were picked, and the DNA purified. The viral DNA was amplified by PCR for the presence of the JNK1-APF cDNA.

Human adenoviruses do not readily infect hematopoietic cells of human or rodent origin, apparently due to the lack of the cellular adenovirus and coxsackievirus receptor (CAR) (44). CAR was introduced into Nb2 cells by infecting the cells with the LXSN retrovirus encoding CAR{Delta}1 (44), and infected cells were selected using 0.5 mg/ml G418 since the LXSN vector contains the neomycin-resistance gene. After selection of G418-resistant cells, the mixed population of cells was infected with recombinant human adenovirus encoding GFP, and 24 h after transduction, GFP-positive cells were selected separated with a fluorescent cell sorter, and separated into 96-well dishes. The sorted cells were expanded and screened again for their ability to be transduced with the GFP-encoding recombinant adenovirus. Clones with the highest rate of infection were chosen for use in these studies. Typically, >95% of the Nb2/CAR{Delta}1 cells could be transduced with the GFP-adenovirus (data not shown).

Nb2/CAR{Delta}1 cells were transduced with varying multiplicities of infection (MOI) ranging from 1–5 virus particles per cell with either Adeno-GFP or Adeno-JNK1-APF in Fisher’s media lacking horse serum for 1 h, after which complete Fisher’s media supplemented with 10% horse serum and 200 ng/ml oPRL was added. Controls for these studies included normal Nb2 cells that could not be infected with recombinant human adenoviruses (data not shown). Expression of the JNK1-APF protein was confirmed by immunoblotting with anti-HA monoclonal antibody.

DNA Fragmentation Assay
Nb2 and Nb2/CAR{Delta}1 were transduced with the adeno-JNK1-APF virus as described above and then cultured for 0–48 h in Fisher’s media supplemented with 10% horse serum and 200 ng/ml oPRL. At 0, 24, and 48 h, the cells were lysed in DNA lysis buffer (50 mM Tris, pH 7.5, 10 mM EDTA, 0.5% SDS, 100 µg RNase A, 4 µg/ml Proteinase K) and incubated at 37 C for 6 h. The DNA was extracted using 400 µl phenol- chloroform-isoamyl alcohol (25:24:1). 0.25 M sodium acetate was added, and the DNA was precipitated using 100% ethanol. The DNA was pelleted and resuspended in TE with 20 µg/ml RNase. The DNA was quantitated spectroscopically, and 10 µg of DNA were analyzed for fragmentation on a 1.5% agarose gel.


    ACKNOWLEDGMENTS
 
The authors thank Karen Helm of the University of Colorado Cancer Center FACS Core for her assistance in isolating the Nb2/CAR{Delta}1 cells. We also thank Drs. Elizabeth Burton, Arthur Gutierrez-Hartmann, and Mary Reyland for their comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Steven M. Anderson, Department of Pathology, Box B-216, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail: steve.anderson{at}uchsc.edu

This research was supported by NIH Grant DK-53858 (to S.M.A.), and a University of Colorado Cancer Center "Wines for Life" Seed Grant. The University of Colorado Cancer Center FACS Core is supported by a Cancer Center grant (CA-46934) from the National Cancer Institute.

{hd2}Note Added in Proof

{texf}While this manuscript was under review, Olazabal et al. (54 ) demonstrated that PRL stimulation of bovine mammary gland epithelial cells transfected with the long from of the PRLR stimulated JNK activation. Furthermore, treatment of these cells with dexamethasone inhibited JNK activated. Although these authors demonstrated that PRL can induce proliferation and AP-1 activation, they do not demonstrate that dominant negative JNK mutants block either of these effects.

Received for publication November 17, 1999. Revision received June 20, 2000. Accepted for publication June 28, 2000.


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