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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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. 1A
, 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. 1B
). Antibody against NKCC detected
a protein of 195 kDa in rat colon and 190 kDa in HC11 cells (Fig. 1B
).
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.
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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. 2A
, 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.
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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. 2B
, 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. 2B
). 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. 2B
). 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. 3
, 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. 3
) 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.
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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
: 340/380 nm; Em
:
505 nm) ratio, a measure of the
[Ca2+]i, was 0.66+0.09
(n = 3) under basal conditions (Fig. 4A
). 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. 4A
). 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).
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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. 4B
, 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. 4C
). 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. 5
). 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.
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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. 6A
, 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).
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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. 6B
, 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. 6C
, 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. 7A
(left panel), CFTR
antibody could not recognize any protein in the immunoprecipitated
samples from HC11 cells (lanes 13), although it recognized the
predicted diffuse bands (175185 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. 7A
, right
panel). This blot was stripped and reprobed with NKCC antibody
(Fig. 7B
). 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. 7
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).
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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. 8A
, 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. 8B
). 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.
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DISCUSSION
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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. 1
and 2
). Both mRNA and
protein for CFTR and NKCC1 expression (Fig. 1
) 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. 1
and 7
). 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 1020 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. 4
), 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
|
---|
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
Scheffes 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 35
for Cl- transport experiments, 2 each for the
experiments described in Figs. 7
and 8
, 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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