Janus Kinase 2 (JAK2) Regulates Prolactin-Mediated Chloride Transport in Mouse Mammary Epithelial Cells through Tyrosine Phosphorylation of Na+-K+-2Cl- Cotransporter

Nataraja G. Selvaraj, Ellen Omi, Geula Gibori and Mrinalini C. Rao

Department of Physiology and Biophysics University of Illinois at Chicago Chicago, Illinois 60612


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Epithelial chloride (Cl-) transport is achieved by the coordinated action of symporters such as the Na+-K+-2Cl- cotransporter (NKCC1) and chloride channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). As a secretory tissue, mammary epithelial cells are obvious candidates for such mechanisms, but Cl- transport and its hormonal regulation have been poorly delineated in mammary epithelial cells. We determined whether the mammary epithelial cell line, HC11, transports chloride and whether this was regulated by PRL, a hormone known to stimulate ion transport. HC11 cells express both CFTR and NKCC1. Exposure to PRL or PGE1 increased Cl- transport in HC11 cells. This was inhibited by the NKCC1 blocker, furosemide, and by the Cl- channel inhibitor, diphenylamine 2-carboxylate. Dose and time course of PRL action indicate that PRL had maximal effect on Cl- transport at 1 µg/ml and at 10 min of stimulation. Examination of the signaling pathways suggests that the PRL effect on Cl- transport does not involve an increase in [Ca2+]i or MAP kinase activity. RT-PCR analyses indicate that HC11 cells express mRNA for Janus kinase 1 (JAK1), JAK2, and signal transducer and activator of transcription 5 (STAT5) but not for JAK3. PRL treatment of HC11 cells increased phosphorylation of STAT5. The JAK2 inhibitor AG490 blocked phosphorylation of STAT5 and PRL-induced, but not PGE1-induced, Cl- transport. NKCC1, but not CFTR, is tyrosine phosphorylated in HC11 cells. PRL enhanced tyrosine phosphorylation of NKCC1, and this effect was attenuated by the JAK2 inhibitor AG490. These results are the first demonstrations of a role for tyrosine phosphorylation of NKCC1 and of the PRL-JAK2 cascade in the regulation of Cl- transport.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fluid movement across epithelia is achieved by the coordinated action of distinct transporters on the apical and basolateral membranes of epithelial cells (1). The Na+/K+ATPase, K+ channels, and Na+-K+-2Cl- cotransporter located on the basolateral membrane with Cl- channels such as CFTR (cystic fibrosis transmembrane conductance regulator) present on the apical membrane function to secrete Cl-, which forms the basis of most fluid secretion (2). In contrast, the basolateral membrane Na+/K+ATPase, in conjunction with Na+ channels or Na+/H+ exchangers on the apical membrane, underlie fluid absorption (2). The coordinated action of both absorptive and secretory processes regulates net fluid balance across most epithelia (1, 2).

Mammary gland is an ideal tissue for studying the regulation of fluid transport because of its necessity to move milk components into the ducts during lactation. While the regulation of mammary milk protein secretion has been widely studied (3), there are few studies examining the regulation of secretion of the accompanying fluid. In lactating rat mammary tissue slices, Cl- transport is mediated by an anion exchange process and via a furosemide-sensitive Na+-K+-2Cl- cotransport (4). BALB/c-mouse mammary epithelial cells grown on floating collagen gels exhibit conductive amiloride-sensitive Na+ transport (5). In addition to being a major lactogenic hormone, the role of PRL as an osmoregulatory hormone in a variety of species is well established. The effects of PRL on stimulating rat intestinal NaCl absorption and increasing Na+ transport have been reported (6) but not characterized. The effects of PRL on Cl- transport are poorly understood. In contrast, the lactogenic effects of PRL leading to mammary gland differentiation and milk protein (ß-casein and whey acidic protein) production have been extensively studied (7, 8).

PRL has pleiotropic actions and utilizes a variety of signaling cascades to elicit this effect. The PRL receptor (PRLR) is a single-transmembrane protein that belongs to the cytokine receptor superfamily (3). Multiple isoforms of PRLR are expressed due to the alternative splicing of the PRLR gene (3). The most extensively studied signaling pathway of PRL is the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. Ligand binding triggers dimerization of the receptor and activation of the receptor-associated JAK2. JAK2 mediates phosphorylation of STAT5, which dimerizes and translocates to the nucleus and brings about the genomic effect of PRL. In addition to JAK-STAT, PRL also stimulates mitogen-activated protein kinase (MAPK) (9, 10) and Fyn, a member of the Src kinase family, in rat T lymphoma Nb2 cell line (11). In human 293 cell line transfected with PRLR, PRL caused a rapid induction of tyrosine phosphorylation of PI-3-kinase and insulin receptor substrate-1 (12). Cross-talk between PRLR and epidermal growth factor (EGF) receptor has also been reported (13). A role for intracellular Ca2+ in PRL action has been implicated in mammary gland explants (14, 15) and in CHO cells transfected with PRLR (16).

Herein we report that PRL regulates Cl- transport in the mouse mammary epithelial cell line, HC11. This is a clonal line derived from the COMMA-1D mouse mammary cell line (7). Treatment of HC11 cells with lactogenic hormones dexamethasone, insulin, and PRL leads to differentiation and production of the milk proteins ß-casein (7) and whey acidic protein (8). In contrast, mouse mammary epithelial cell lines like NOG-8 do not respond to lactogenic hormones to stimulate milk protein production (8). Thus, HC11 serves as a good in vitro model with which to study the growth and differentiation of mammary cells (17). We show that HC11 cells express mRNA and protein for CFTR and NKCC1, an isoform of the Na+-K+-2Cl- cotransporter. PRL and PGE1 increase Cl- transport in these cells. PRL-mediated Cl- transport is not through an increase in intracellular Ca2+ or by increased MAPK activity. However, blocking JAK2 with AG490 can specifically inhibit PRL-mediated Cl- transport and STAT5 phosphorylation. We observed that NKCC1, but not CFTR, is tyrosine phosphorylated, and PRL treatment results in an increase in tyrosine phosphorylation of NKCC1, while AG490 decreases it. This is the first demonstration of a role for the PRL-JAK2 cascade in regulating epithelial Cl- transport.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Ion Transporters
We first determined whether mammary epithelial cells express CFTR and Na+-K+-2Cl- cotransporters required for Cl- transport. The primer for CFTR was designed from the mouse cDNA sequence (18). Two distinct but highly homologous isoforms of Na+-K+-2Cl- cotransporter exist in mammalian tissue (19). The secretory form of the cotransporter, NKCC1, is localized to the basolateral membrane of secretory epithelia, while NKCC2 is present in the apical membrane of salt-absorptive epithelia such as the thick ascending loop of Henle (19). Primers for NKCC1 were generated based on human cDNA sequences (20) and for NKCC2 from the published rabbit sequence (21). This and all other RT-PCR assays described in this paper were qualitative analyses. We used rat, mouse, and human colon samples as positive controls for NKCC1 and CFTR and rabbit kidney as positive control for NKCC2. As shown in Fig. 1AGo, mouse proximal colon and HC11 cells possess the transcript for CFTR. While NKCC1 was expressed in both HC11 cells and the human colonic cell line, T84, NKCC2 mRNA was present in rabbit kidney but not in HC11 cells. To confirm the presence of proteins for the transporters, immunoblot analysis was carried out using membrane isolated from HC11 cells. The CFTR antibody detected a 185-kDa protein in T84 and a single prominent 175-kDa protein in HC11 cells (Fig. 1BGo). Antibody against NKCC detected a protein of 195 kDa in rat colon and 190 kDa in HC11 cells (Fig. 1BGo). Both CFTR and NKCC are glycoproteins and therefore often appear as diffuse bands on Western blots (also see Discussion).



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Figure 1. Expression of CFTR and NKCC1 in HC11 Cells

A, Total RNA isolated from HC11, T84 cells, rabbit kidney, and mouse colon were reverse transcribed and PCR was carried out using specific primers as explained in Materials and Methods. B, Western blot analysis of membrane proteins. The samples were resolved by SDS-PAGE on 6% gel, after which the protein was transferred to nitrocellulose membrane. The membrane was probed with antibody specific to CFTR or NKCC. Please note that both CFTR and NKCC are glycosylated proteins. HC, HC11 cells; RK, rabbit kidney; MC, mouse colon; T84, human colonic cell line; RC, rat colon.

 
Expression of PRLR and Regulation of Cl- Transport in HC11 Cells
Since PRL can signal through at least two types of receptors (3), we examined, by RT-PCR, PRLR expression in the HC11 cells. As shown in Fig. 2AGo, HC11 cells express mRNA for the long form of the PRLR but not for the short form, suggesting that PRL-mediated stimulation of Cl- influx in HC11 cells occurs through the PRLR long form.



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Figure 2. Expression of PRLR (A) and Regulation of Cl- Transport in HC11 Cells (B)

A, RT-PCR analysis of RNA from HC11 (HC) and rat liver (RL). Total RNA was subjected to reverse-transcriptase followed by amplification using specific primers for the long form or the short form of the receptor. B, Cells were loaded with MQAE, transferred to Cl- and HCO3- free medium and incubated with either DPC (50 µM) or furosemide (10 µM) alone or together either in the presence or absence of 1 µg/ml PRL or 1 µM PGE1 for 5 min at room temperature. Cl- influx was calculated as indicated in Materials and Methods. Data are mean ± SEM, n = 4, where each "n" represents an average of triplicate determinations *, P < 0.01, ANOVA.

 
Chloride transport was measured using the halide-sensitive fluorescent dye MQAE. The fluorescence of MQAE is quenched by nonphysiological anions such as thiocyanate and nitrite and weakly by other intracellular anions. To account for this, the Stern-Volmer constant (KCl) was first determined for HC11 cells, and it was found to be 98 M-1. Chloride transport in HC11 cells was characterized using specific inhibitors to block the activity of the cotransporter and CFTR. Furosemide has been demonstrated to block NKCC-cotransporter activity, while the CFTR Cl- channel can be inhibited by diphenyl amine 2-carboxylate (DPC) (22). We have previously reported that neither of these inhibitors interfere with MQAE fluorescence (22). As can be seen in Fig. 2BGo, basal Cl- permeability (0.57 ± 0.03 mM/sec) was inhibited by both DPC (0.45 ± 0.03 mM/sec) and furosemide (0.43 ± 0.05 mM/sec), either treated alone or together (0.43 ± 0.03 mM/sec). These data further confirm the presence of functional Cl- channels and the Na+-K+-2Cl- cotransporter. PRL caused a significant increase in Cl- influx (0.86 ± 0.07 mM/sec), which was blocked by DPC (0.52 ± 0.03 mM/sec) or furosemide (0.49 ± 0.04 mM/sec) either added alone or together (0.38 ± 0.03 mM/sec) (Fig. 2BGo). This suggests that Cl- transport induced by PRL is through regulating the Cl- channel and Na+-K+-2Cl- cotransporter. PGE1 at a dose of 1 µM stimulated Cl- influx (0.84 ± 0.04 mM/sec), and the Cl- permeability mediated by PGE1 was also inhibited by DPC (0.51 ± 0.06 mM/sec) or furosemide (0.52 ± 0.07 mM/sec) added alone or in combination (0.49 ± 0.07 mM/sec) (Fig. 2BGo). The Cl- permeability in the presence of inhibitors, i.e. inhibitor-insensitive transport, was similar in basal, PRL-, and PGE1-treated cells.

Dose and Time Course of PRL Response
As shown in Fig. 3Go, PRL caused a significant increase in Cl- influx (mM/sec) at 0.1 µg/ml (0.92 ± 0.08), while it had no effect at lower doses of 0.01 µg/ml (0.73 ± 0.07) and 0.001 µg/ml (0.67 ± 0.02) compared with the basal (0.62 ± 0.04). At 1 µg/ml of PRL, the response was maximal (1.01 ± 0.03), and there was no further increase at a higher dose of 10 µg/ml (0.95 ± 0.09). The EC50 for the dose response was observed to be 0.08 µg/ml PRL. In the presence of DPC and furosemide, Cl- influx was decreased both in the basal as well as in PRL-induced conditions. Time course of PRL mediated Cl- influx in HC11 cells (Fig. 3Go) revealed no significant increase in Cl- secretion within 2 min of PRL treatment. However, a significant increase was seen by 5 and 10 min of PRL exposure. The effect of PRL on Cl- transport declined over time, reaching baseline levels by 60 min. The inhibitors, DPC and furosemide added together, were able to inhibit the Cl- influx at all time points.



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Figure 3. Dose and Time Course of PRL Action on Chloride Influx in HC11 Cells

HC11 were loaded with MQAE, Cl- depleted, and stimulated with PRL either in the absence or presence of DPC and furosemide for 5 min or for varying time at room temperature. Rate of change of fluorescence after addition of NaCl was monitored, and Cl- influx was calculated as explained in Materials and Methods. A, Cells were incubated either in the absence or with increasing concentrations of PRL for 5 min, and Cl- influx was measured. B, Cells were incubated either in the presence or absence of 1 µg/ml PRL for various time and Cl- influx monitored. Values are mean ± SEM; n = 5 for panel A and n = 4 for panel B; *, P < 0.01, ANOVA.

 
PRL Signaling in HC11 Cells
Because the transient nature of PRL-mediated Cl- transport is reminiscent of Ca2+-induced Cl- secretion in other epithelial cells (23) and since PRL can increase intracellular Ca2+ in some cell types (15, 16), we examined the effects of PRL on [Ca2+]i in HC11 cells using the Ca2+-sensitive fluorescent dye Fura-2 AM. The relative fluorescence (Ex{lambda}: 340/380 nm; Em{lambda}: 505 nm) ratio, a measure of the [Ca2+]i, was 0.66+0.09 (n = 3) under basal conditions (Fig. 4AGo). While the Ca2+ ionophore A23187 significantly increased the ratio (1.63 ± 0.32, n = 3), PRL at 1 µg/ml caused no change in [Ca2+]i (ratio: 0.73 ± 0.32, n = 3, Fig. 4AGo). As shown by others, A23187 did not interfere with Fura-2 fluorescence (24).



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Figure 4. Measurement of Intracellular Ca2+ and Activation of MAPK in HC11 Cells

A, Cells were loaded with Fura-2AM for 30 min and [Ca2+]i measured by excitation at 340/380 nm and emission at 505 nm. The basal fluorescence was measured first followed by addition of PRL (1 µg/ml) and the Ca2+ ionophore A23187 (1 µM) as described in Materials and Methods. B and C, The cells were stimulated with either PRL (1 µg/ml) or EGF (100 ng/ml) for 10 min at 37 C and harvested, and membrane and cytosolic fractions were prepared. The cytosol fraction was resolved by SDS-PAGE on 9% gel, followed by electrotransfer to nitrocellulose membrane. The blot was probed with phospho-ERK antibody (B) and visualized using ECL reagent after incubating with secondary antibody conjugated with peroxidase. The blot was stripped and reprobed with anti-pan ERK antibody (C).

 
PRL was reported to activate MAPK in human leiomyoma and rabbit mammary cells (9, 10), so the involvement of this pathway in PRL-mediated Cl- transport in HC11 cells was next examined. As shown in Fig. 4BGo, Western blot analysis of the cytosol of HC11 demonstrated that EGF treatment of HC11 cells increased phosphorylation of ERK1 and ERK2 seen at 44 and 42 kDa, respectively (lane 3). However, in the presence of PRL (lane 2) or in the basal condition (lane 1), there was no phosphorylation of ERK. Reprobing this blot with pan ERK antibody (which recognizes both phosphorylated and nonphosphorylated ERK) indicates that ERK protein is expressed at comparable levels in all samples (Fig. 4CGo). These data suggest that, in HC11 cells, EGF stimulated the phosphorylation of MAPK, while PRL had no effect.

JAK-STAT Signaling
The major signaling pathway linked to PRL action is the JAK-STAT system (3). Therefore, expression of this system was examined by RT-PCR analysis in HC11 cells. Specific messages of expected sizes were amplified for JAK1, JAK2, and STAT 5 in HC11 cells, but mRNA for JAK3 was absent (Fig. 5Go). However, GGAD, a cell line derived from rat decidual cells express JAK1, JAK2, STAT5 (25), and JAK3 (our unpublished observation). Ample expression of GAPDH in all samples ensured the quality of the RNA.



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Figure 5. Expression of JAK-STAT System in HC11 Cells

RNA isolated from HC11 cells or the rat decidual cell line GGAD was reverse transcribed to first-strand cDNA followed by PCR for 25 cycles with specific primers as described in Materials and Methods. Lane 1, HC11; lane 2, GGAD cells.

 
Effect of Blocking JAK2 Activity
To determine whether JAK2 is involved in PRL-mediated Cl- transport, the effects of AG490, an agent known to inhibit JAK2 activity, on PRL action in HC 11 cells was examined. As a control, the cells were also stimulated with PGE1, which is known to activate Cl- transport by increasing [cAMP]i (22). As shown in Fig. 6AGo, inhibition of JAK2 activity with AG490 (50 µM) had very little effect on the basal level (inhibitor-sensitive Cl- transport). Whereas both PRL and PGE1 increased Cl- permeability, only PRL-mediated increase in Cl- influx was prevented by AG490. The JAK2 inhibitor had no effect on PGE1-induced Cl- influx.



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Figure 6. Effect of PRL and AG490 upon Cl- Influx and STAT5 Phosphorylation in HC11 Cells

A, Cells were loaded with MQAE for 90 min on ice either in the presence of AG490 (50 µM) or an equal volume of DMSO, followed by transfer of the cells to Cl- free buffer ± AG490 (50 µM) for 30 min. The cells were incubated ± PRL (1 µg/ml) or PGE1 (1 µM) and ± inhibitors (DPC and furosemide) for 5 min and Cl- flux calculated as indicated in Materials and Methods. The data represent the inhibitor-sensitive Cl- transport (net Cl- transport obtained after subtracting the Cl- influx in the presence of DPC and furosemide) and is mean ± SEM, n = 4, where each n is the average of triplicate determinations; a, P < 0.05 compared with control; b, P < 0.01 compared with PRL alone, ANOVA. B and C, Phosphorylation of STAT5 by PRL in HC11 cells. The cells were preincubated with AG490 (50 µM) for 2 h in serum-free media, followed by stimulation with PRL (1 µg/ml) or EGF (100 ng/ml) for 10 min. The cells were lysed and immunoprecipitated with a polyclonal antibody against STAT5. The immunoprecipitate was resolved in 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with a monoclonal antibody against phospho-STAT5 (B). The blot was stripped and reprobed with STAT5 antibody (C).

 
Activation of STAT5 by JAK2
To demonstrate biochemically that JAK2 is activated upon stimulation with PRL, the phosphorylation of STAT5 was examined in HC11cells. The cells were preincubated in serum-free medium for 2 h with or without AG490, followed by stimulation with PRL or EGF, and the cell lysates were immunoprecipitated with STAT5 polyclonal antibody. The immunoprecipitate was resolved in 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with a monoclonal antibody against phospho-STAT5. As can be seen in Fig. 6BGo, treatment of the cells with PRL (1 µg/ml) stimulated the phosphorylation of STAT5. AG490 reduced significantly the phosphorylation induced by PRL. EGF was able to stimulate STAT5 phosphorylation, but to a much lesser extent compared with PRL. To examine whether this difference in the phosphorylated state of STAT5 was due to change in the protein level, the blot was stripped and reprobed with STAT5 antibody. As shown in Fig. 6CGo, all samples expressed the protein at comparable levels.

NKCC1 but Not CFTR Is Tyrosine Phosphorylated
Since JAK2 is a tyrosine kinase, we examined whether CFTR and/or NKCC1 are phosphorylated on tyrosine residues. Cells were stimulated with PRL (1 µg/ml) or EGF (100 ng/ml) for 10 min, lysed, and immunoprecipitated with phosphotyrosine antibody. The immunoprecipitate and the supernatant were subjected to SDS-PAGE on 6% gels, transferred to nitrocellulose membrane, and probed with CFTR antibody. As shown in Fig. 7AGo (left panel), CFTR antibody could not recognize any protein in the immunoprecipitated samples from HC11 cells (lanes 1–3), although it recognized the predicted diffuse bands (175–185 kDa) in the membrane isolated from HC-11 (lane 5) and T-84 cells (lane 4). In contrast, the supernatant of the immunoprecipitated samples contained CFTR (Fig. 7AGo, right panel). This blot was stripped and reprobed with NKCC antibody (Fig. 7BGo). The presence of NKCC in the precipitate indicates that this protein is tyrosine phosphorylated. However, the supernatant consistently showed two bands when analyzed with NKCC antibody; the nature of the lower band is not known and could be either a proteolytic or a differentially glycosylated product of the protein. The presence of NKCC in the supernatant suggests that either the immunoprecipitation (IP) of tyrosine-phosphorylated proteins was incomplete or that not all of the NKCC pool is tyrosine phosphorylated. To examine whether IP was complete, the precipitate and the supernatant blots were probed with phosphotyrosine antibody. While the precipitate blots revealed multiple bands, that of the supernatant had none (data not shown), indicating that IP with antiphosphotyrosine was complete. Densitometric scanning of the immunoprecipitate lanes, probed with NKCC antibody, showed a 30% increase in density of the protein in PRL-treated samples as compared with the basal or EGF samples. This modest increase may be a consequence of the experimental design: the antiphosphotyrosine immunoprecipitate is probed with NKCC antibody, which assesses the amount of the protein rather than its degree of phosphorylation. Therefore, if PRL stimulation renders an increase in the number of phosphotyrosine residues/NKCC molecule more than an increase in the number of NKCC proteins phosphorylated, it will not be readily detected by this protocol. Thus, although tyrosine phosphorylation of NKCC may be increased, we could quantitate this as a modest change in signal when the antiphosphotyrosine immunoprecipitate was probed with NKCC antibody. In summary, the data in Fig. 7Go indicate that in HC11 cells a fraction of the cellular NKCC is tyrosine phosphorylated, and this appears to be regulated by PRL.



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Figure 7. NKCC1 but Not CFTR Is Tyrosine Phosphorylated

The cells were lysed after stimulation with PRL (1 µg/ml) or EGF (100 ng/ml) for 10 min and immunoprecipitated with a monoclonal antibody against phosphotyrosine. The immunoprecipitate (Ppt), supernatant (Sup.), and membrane proteins from HC11 and T84 cells were resolved by SDS-PAGE on 6% gels, transferred to nitrocellulose, and probed with a polyclonal antibody against CFTR (A). The blots were stripped and reprobed with NKCC1 antibody (B).

 
PRL Increases Tyrosine Phosphorylation of NKCC1
To confirm that PRL was indeed altering NKCC phosphorylation and since phosphotyrosine antibodies do not immunoprecipitate the entire pool of NKCC, we used the converse approach. We also determined whether the JAK2 inhibitor AG 490 blocked this effect. Cells, treated with buffer, PRL ± AG 490, were first immunoprecipitated with anti-NKCC antibody and then subjected to Western analysis using phosphotyrosine antibody. In data not shown, we observed that the IP with NKCC antibody was complete, there being no detectable NKCC protein in the supernatant. As shown in Fig. 8AGo, PRL increased tyr-phosphorylation of the immunoprecipitated NKCC (lane 3) as compared with control (lane 1). This increase in phosphorylation is inhibited by pretreating the cells with AG490 (lane 4). In contrast, EGF had no effect on tyrosine phosphorylation of NKCC1 (lane 5). To normalize the samples for the amount of NKCC, the blots were stripped and reprobed with NKCC antibody (Fig. 8BGo). Both the antiphosphotyrosine and anti-NKCC blots were subjected to PhosphorImager analysis. There was relatively little variability between the samples in the NKCC blot (arbitrary units): basal, 1.0; AG490, 1.10; PRL, 0.99; PRL+AG490, 1.09; and EGF, 1.10. These values were used to normalize the quantitation of the blot in panel A. The results are expressed as fold-stimulation over basal and shown in panel C. PRL treatment causes a 4.13-fold increase in tyrosine phosphorylation of NKCC1 in HC11 cells, and this increase was inhibited in the presence of AG490.



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Figure 8. JAK2 Regulates PRL-Mediated Tyrosine Phosphorylation of NKCC1

The cells were preincubated with AG490 (50 µM) for 2 h in serum-free condition, followed by stimulation with PRL (1 µg/ml) or EGF (100 ng/ml) for 10 min. The cells were lysed and immunoprecipitated with a monoclonal antibody against NKCC. The immunoprecipitate was resolved by SDS-PAGE on 6% gels, transferred to nitrocellulose, and probed with a monoclonal antibody against phosphotyrosine (panel A). This blot was subjected to PhosphorImager analysis for quantitation. To normalize the samples for the amount of NKCC, the blots were stripped and reprobed with NKCC antibody, and this blot was also subjected to Phosphorimager analysis. The variability between the samples in the NKCC blot was <5% (panel B). The antiphosphotyrosine values were normalized for NKCC content and the results expressed as fold stimulation over basal. The data shown in panel C are the mean of two experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the current study, we have characterized HC11 cells as a model for epithelial cells of mammary gland to study Cl- transport. We have provided the first demonstration that these cells possess the machinery required for Cl- transport (Figs. 1Go and 2Go). Both mRNA and protein for CFTR and NKCC1 expression (Fig. 1Go) indicate that this cell line is an ideal candidate to study Cl- transport. CFTR and NKCC1 are glycoproteins known to show differences in relative mobility, in band width and in the diffuseness of the band on SDS-PAGE, depending on the tissues and/or species examined (26, 27). They can also exist as differentially glycosylated forms within a cell type (26, 27). Thus, the variability in the relative molecular mass of these proteins in HC 11 cells and those of the positive control are not unusual (Figs. 1Go and 7Go). The presence of DPC- and furosemide-sensitive Cl- transport confirms the functional expression of these transport proteins in HC11 cells. This is in marked contrast to the murine mammary epithelial cell line, C127, which lacks the expression of CFTR (28). Studies in rat mammary epithelial slices indicate that they possess a furosemide-sensitive cotransporter (29). Recent studies have demonstrated that mammary glands also express Ca2+-sensitive chloride channels (30), but in none of these studies has regulation by PRL been investigated.

Multiple isoforms of PRLR have been identified in several species (3). In mouse, at least four forms of PRLR expressed in different tissues have been identified (31), while the rat has two isoforms (3), termed long and short form. The long form of the receptor is believed to mediate most of the known biological effects of PRL, while the actions ensuing from the activation of the short form of PRLR remain to be elucidated. However it has been shown that PRLR-associated protein (PRAP) preferentially interacts with the short PRLR in rat corpus luteum (32). The current study has demonstrated that, as in mouse mammary gland (31), HC11 cells express the long form but not the short form of PRLR.

PRL showed a dose-dependent effect on Cl- transport with a maximal effect at 1 µg/ml. These concentrations were also found to stimulate maximally ß-casein gene expression in these cells (7). Induction of Cl- transport in HC11 cells could be blocked by either furosemide and/or DPC. These drugs have been used extensively to characterize the Cl- transport machinery in other epithelia (33). The lack of complete inhibition of Cl- transport in the presence of DPC and furosemide may be due to the expression of Ca2+-sensitive chloride channels that are not blocked by these inhibitors. However, the effect of PRL appears to be mediated predominantly by the DPC sensitive Cl- channels since PRL has very little effect on the inhibitor-insensitive component of the Cl- transport. The effect of PRL on Cl- transport appears to be transient with the increase in Cl- influx being maximal between 5 and 10 min, after which it declines. It is well documented in a number of epithelia that Ca2+-dependent agents have a transient effect on Cl- transport (2, 23).

Evidence for the presence of Cl- transport in mammary epithelial cells is not clear. While it has been suggested that there is no or little active Cl- transport in mammary epithelial cell cultures (5, 34), other studies have indicated the presence of both paracellular and transcellular movement of Cl- (4, 35). Our study clearly suggests that the mammary epithelial cells, as exemplified by HC11 cells, are capable of transporting Cl-. In terms of ion transport regulation, prolonged (>= 3 days) exposure to PRL was shown to increase potential difference and short circuit current (Isc), in mouse mammary epithelial cells on floating collagen gels (5). The increase in Isc, an index of ion transport, was in large part due to sodium absorption and PRL did not significantly alter net Cl- movement (5). This is in contrast to the present study where we observe that PRL causes a rapid and transient increase in Cl- transport in the mouse mammary epithelial cell line HC11. In the earlier study, only long-term (>= 3 days) effects of PRL were examined and could account for this difference. Our study also demonstrates that PGE1, known to act via cAMP in a variety of epithelia (33), also stimulates Cl- transport in HC11 cells.

Multiple signaling pathway(s) have been implicated in PRL action. The time course of PRL-mediated chloride transport is reminiscent of the effects of Ca2+-dependent secretagogues on Cl- transport in intestinal epithelia (23). However, PRL had no effect on [Ca2+]i, suggesting that, in HC11 cells, PRL does not utilize the Ca2+ signaling pathway. PRL also has a rapid and transient effect on MAPK activity in some systems. Thus, intraperitoneal administration of PRL to rats increased hepatic MAPK activity within 5 min, sustained activation for 10–20 min, and returned it to basal level by 30 min (36). In mouse mammary epithelial cell line, NOG-8, PRL also increased MAPK activity (37). However we show that in HC11 cells EGF, but not PRL, stimulates MAPK activity (Fig. 4Go), and this is not inhibited by AG490 (data not shown).

JAK2 is the major PRLR-associated JAK (3). STAT5 was initially described as mammary gland factor stimulated by PRL (38). Gene deletion studies of STAT5 clearly indicate the importance of the JAK/STAT pathway for PRL signaling (39, 40). The presence of JAK2 and STAT5 transcripts in HC11 cells and the earlier demonstration that PRL induction of ß-casein gene expression involves this pathway in HC11 cells suggest that this JAK2/STAT5 might mediate PRL action on Cl- transport (38). Our studies show that PRL increased phosphorylation of STAT5 in HC11 cells, indicating that JAK2 is activated by PRL. This is consistent with the earlier observation that PRL regulates STAT5 phosphorylation and nuclear translocation in HC11 cells (41). Using the JAK2 inhibitor, AG490 (42), we demonstrate in this paper that AG490 blocks STAT5 phosphorylation as well as PRL-mediated, but not PGE1-stimulated, Cl- transport. This indicates that PRL brings about its effect on Cl- transport through the JAK2 signaling cascade. The transient effect of PRL suggests that the action of JAK2 on Cl- transport is not through the conventional genomic pathway but through a more rapid, perhaps nonnuclear, cytosolic/membrane effect on the transporters.

Activation of JAK2 by PRL leads to phosphorylation of signaling molecules other than STAT5, including insulin receptor substrate (43) and SHC protein that recruits GRB2-SOS complex (44). The activity of CFTR and NKCC1 are known to be modulated by their phosphorylation, and, depending on the system, increased phosphorylation of specific Ser/Thr residues is associated with increased activity (45, 46). Functionally we observe that an inhibitor of NKCC (furosemide) as well as a chloride channel blocker (DPC) inhibited PRL-mediated Cl- transport, implicating both transporters as targets of PRL action. However, we demonstrate that only NKCC1, but not CFTR, is tyrosine phosphorylated and this is increased in response to PRL. Furthermore, this PRL-induced tyrosine phosphorylation of NKCC1 is inhibited by AG490. This is the first demonstration that NKCC1 is tyrosine phosphorylated and that this phosphorylation is specifically increased by JAK2. In cerebral microvessel endothelial cells, NKCC expression, phosphorylation, and activity were increased by astrocytes conditioned media, and this effect was demonstrated to be through IL-6 (47, 48). While IL-6 is known to utilize the JAK-STAT pathway (49), increased tyrosine phosphorylation of NKCC in endothelial cells was not examined (45, 46). Our data clearly suggest that JAK2 causes an increase in tyrosine phosphorylation of NKCC. It remains to be determined whether this results from a direct association of the kinase and the transporter or if it involves an intermediate protein(s). If the former, the association is not very tight, since IP of control, PRL-treated, or EGF-treated cells with anti-NKCC failed to coprecipitate JAK2 (data not shown). In addition to NKCC, the Cl- transport studies suggest that PRL can affect DPC-sensitive transport and therefore CFTR function. Clearly this is not via tyrosine-specific phosphorylation of CFTR and must invoke intermediary, as yet unknown, signaling steps.

In conclusion, we present evidence for the first time that the mammary epithelial cell line, HC11, exhibits characteristics of chloride-secreting cells expressing CFTR and NKCC1. We further report that PRL increases Cl- transport in these cells through the long form of the PRLR and the JAK/STAT system. We have also established for the first time that NKCC1 is tyrosine phosphorylated, and PRL increases tyrosine phosphorylation of NKCC1 through JAK2. The HC11 cell line could be useful for studying PRL regulation of both the exocrine protein and fluid secretory functions of mammary epithelia.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
RPMI-1640, FBS, antibiotics, trypsin, and reagents used for RT-PCR were procured from Life Technologies, Inc. (Gaithersburg, MD). 32P-CTP and enhanced chemiluminescence (ECL) reagent were from Amersham Pharmacia Biotech (Piscataway, NJ). Fluorescent probe 6-methoxyquinolylacetylethyl ester (MQAE) and FURA-2AM were from Molecular Probes, Inc. (Eugene, OR). Insulin, EGF, protease inhibitors, and other reagents were of analytical grade and obtained from Sigma (St. Louis, MO). The sources of antibodies are as follows: antiphosphotyrosine monoclonal antibody and STAT5 polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antiphospho-ERK1/2 and antiphospho-STAT5 were from New England Biolabs, Inc. (Beverly, MA); and anti-pan ERK was from Transduction Laboratories, Inc. (Lexington, KY). Antibodies against CFTR and NKCC were generous gifts of Dr. A. Nairn (The Rockefeller University, New York, NY) and Dr. C. Lytle (University of California, Riverside, CA), respectively. Ovine PRL (AFP-10677C) was a gift from NIDDK (NIH, Bethesda, MD).

Cell Culture
HC11 cells were grown in RPMI-1640 media containing 10% FBS, insulin (5 µg/ml), EGF (10 ng/ml), and the antibiotics ampicillin (25 µg/ml), streptomycin (270 µg/ml) and Amphotericin (1.25 µg/ml) at 37 C in a CO2 incubator. Confluent cells were detached by incubating with trypsin at 37 C for 4 min. Suspended cells were used for chloride transport studies.

Chloride Transport
Chloride transport was measured using the halide-sensitive fluorescence probe MQAE as described earlier (22, 33, 50). Suspended cells were loaded with the dye in buffer A containing (in millimolar concentration) 5 MQAE, 110 NaCl, 1 MgCl2, 1 CaCl2, 5 dextrose, 50 mannitol, and 1 KCl (pH 7.4) for 90 min at 4 C. The cells were then suspended in a Cl- and HCO3-free buffer B containing (in millimolar concentration) 110 sodium isethionate, 1 MgSO4, 5 dextrose, 50 mannitol, 1 K2SO4, and 1 CaSO4 (pH 7.4) for 90 min at 4 C. Fluorescence was measured in a PTI spectrofluorometer (Princeton, NJ) at an excitation wavelength of 355 nM and an emission wavelength of 460 nM. Cells were incubated ± stimulants and ± inhibitors (50 µM diphenylamine 2-carboxylate and 10 µM furosemide) for 5 min or at different time points, and the change in fluorescence after the addition of 5 mM NaCl was measured. Background fluorescence was obtained by adding 150 mM KSCN. Cl- influx was calculated using the formula Jcl = (F0/KClF2)(dF/dt), where Jcl is the rate of Cl- influx (mM/sec), dF/dt is the slope of the initial rate of change of fluorescence upon addition of Cl-, KCl is the Stern-Volmer constant for quenching intracellular MQAE by Cl-, and F0 and F are absolute fluorescence in the absence and presence of Cl-, respectively. The background fluorescence was deducted from both F0 and F. KCl was determined for the HC11 cells by quenching the MQAE fluorescence with the addition of increasing concentrations of Cl-. Stern-Volmer constant (KCl) is the slope of the equation F0/F= 1+KCl[Cl-], where [Cl-] is the Cl- concentration. We had previously demonstrated that inhibitors such as furosemide and DPC do not interfere with MQAE fluorescence (22). In addition, every agent tested was first checked for any interference with MQAE fluorescence. None of the relevant agents used in this study interfered with MQAE fluorescence.

Intracellular Ca2+ Measurement
The cells in suspension were loaded with acetoxymethyl ester of fura-2, fura-2AM (2 µM) in 1 ml buffer containing (in millimolar concentration) NaCl 110, KCl 5, dextrose 5, K2(S2O5) 5, Tris 10, Mannitol 30, and CaCl2 0.1, 50 µl FBS, 20% pluronic F-127 for 30 min at 37 C. The cells were centrifuged and suspended in the same buffer with 1 mM CaCl2. Fluorescence was monitored in PTI spectrofluorometer with excitation at 340 and 380 nM and emission at 505 nM. After the basal fluorescence measurement, PRL (1 µg/ml) was added and the measurement continued for 5 min followed by the addition of the ionophore A23187. This inonophore did not interfere with Fura-2 fluorescence.

RNA Isolation and RT-PCR
Total RNA was isolated by a one-step extraction method using the TRIzol isolation kit (Life Technologies, Inc.). RT-PCR was carried out as described earlier with slight modification (33). In brief, total RNA (5 µg) was reverse transcribed to first-strand cDNA with 200 U of Moloney murine leukemia virus reverse transcriptase (MMLV-RT) in the presence of 0.5 mM deoxynucleotide triphosphates (dNTPs) and 25 µg/ml oligo (dt) in a total volume of 20 µl for 60 min at 40 C. After the reaction, 80 µl water was added to the samples and heated at 95 C for 5 min. PCR was performed in a total volume of 50 µl with 5 µl reverse-transcribed product, 2.5 U Taq-polymerase, 50 µM dNTPs, 0.6 µM primers in 1x PCR buffer with 0.025 µCi of 32P-dCTP. Amplification was performed using 1 cycle at 95 C for 2 min followed by 25 cycles at 95 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec and the final extension at 72 C for 5 min (Hybaid, Middlesex, UK). The PCR product was resolved in PAGE, and the radioactivity monitored after exposing the gel to x-ray film (Kodak, Rochester, NY). In all cases the RT-PCR analyses are qualitative. Primers used in PCR were designed from the coding region of various cDNAs using the software Lasergene (DNASTAR, Madison, WI). The primers used were as follows, with the expected product size in parenthesis: CFTR (444) sense 5'-TGC GTA CTG GTT TTT CTG GTT GAG, antisense 5'-GGG TTG TAA TGC CGA GAC GAC TAT; NKCC1 (468) sense 5'-GGC TGG ATC AAG GGT GTA TTA, antisense-5'-ATC GGG CCC AAA GTT CTC ATT; NKCC2 (350) sense 5'- ACA GGA AAG ACT GGC ATT GG, antisense 5'- GGA GGG AAG ATA AAT CGC AT. Sense primer for PRLR was the same for both the long and the short form 5'-AAA GTA TCT TGT CCA GAC TCG CTG, while the antisense primer for the long form was 5'-AGC AGT TCT TCA GAC TTG CCC TT and for the short form was 5'-TTG TAT TTG CTT GGA GAG CAG T. The expected product size was 279 bp for both forms. JAK1 (275), sense 5'-CTA TGA GCC AGC TGA GTT TCG ATC, antisense 5'-CAT CTC GGA CAC AGA CGC CGT A; JAK2 (523), sense 5'-GTT CTT ACC GAA GTG CGT GCG A, antisense, 5'-GGT AAT GGT GTG CAT CGC AGT T; JAK3 (700), sense 5'-CCA GGA AGC TGG AAC GCT CAA C, antisense 5'-CGA ACA GCA GTA GGC GGT GGT T; STAT5 (610), sense 5'-GGG CAT CAC CAT TGC TTG GAA, antisense 5'-CAC GAC TAG TAT TAA CAC TTC AC.

IP
Cells were cultured in 60-mm dishes to confluency and preincubated in serum free media for 2 h either in the presence or absence of JAK2 inhibitor AG490 (50 µM), followed by stimulation with PRL (1 µg/ml) or EGF (100 ng/ml) for 10 min. After stimulation, the plates were transferred to ice and washed thrice with ice-cold PBS, and the cells were scraped in 200 µl lysis buffer (0.15 M NaCl, 50 mM HEPES, pH 7.2, containing 2.5 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 10 µg/ml leupeptin, 30 mM sodium pyrophosphate, 30 mM sodium fluoride, 1 mM orthovanadate, 10% glycerol, and 1% NP40). The cell lysate was sonicated for 10 sec on ice. IP of STAT5 and phosphotyrosine proteins were carried out essentially as reported earlier with slight modifications (51). In brief, the cell lysates were incubated with primary antibody overnight at 4 C in a shaker. The immunoprecipitate was isolated by complete adsorption to a large excess of protein A/G-Sepharose matrix. IP of NKCC was carried out as described by Lytle (46). The adsorbed immunoprecipitate was released from the matrix by heating the sample for 10 min at 60 C with Laemmli buffer and centrifuged, and the supernatant was stored at -20 C until used for Western blotting.

Western Blotting
Membrane protein (50 µg) or immunoprecipitates were subjected to Western blotting as described earlier (52). Briefly, the proteins were separated by SDS-PAGE and the samples electrotransferred to nitrocellulose paper. The immobilized proteins were probed with specific antibody, followed by second antibody conjugated with horseradish peroxidase. The proteins were visualized using ECL reagent. The blots were stripped and reprobed as described elsewhere (41).

Statistics
Statistical significance was calculated using ANOVA followed by Scheffe’s test. Values of P < 0.05 were considered significant. In each experiment the measurements were done in triplicate and averaged to give an n = 1. The n values were 3–5 for Cl- transport experiments, 2 each for the experiments described in Figs. 7Go and 8Go, and at least 3 for all the other experiments.


    ACKNOWLEDGMENTS
 
We are grateful to Drs. C. Lytle and A. Nairn for their kind gift of antibodies against NKCC and CFTR, respectively. We would like to acknowledge the technical help of Dr. S. A. K. Choudhury and Ms. B. Stephan. We thank Ms. J. Gentry for secretarial help.


    FOOTNOTES
 
Address requests for reprints to: Mrinalini C. Rao Ph. D., Department of Physiology & Biophysics (M/C 901), University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Illinois 60612. Email: meenarao@uic.edu.

This work was supported by the NIH Grants DK-46910 (M.C.R.), HD-12356, and HD-11119 (G.G.).

Received for publication May 18, 2000. Revision received August 14, 2000. Accepted for publication August 29, 2000.


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