ArticlePDF Available

Enhanced DNA-Binding Activity of a Stat3-Related Protein in Cells Transformed by the Src Oncoprotein


Abstract and Figures

Cytokines and growth factors induce tyrosine phosphorylation of signal transducers and activators of transcription (STATs) that directly activate gene expression. Cells stably transformed by the Src oncogene tyrosine kinase were examined for STAT protein activation. Assays of electrophoretic mobility, DNA-binding specificity, and antigenicity indicated that Stat3 or a closely related STAT family member was constitutively activated by the Src oncoprotein. Induction of this DNA-binding activity was accompanied by tyrosine phosphorylation of Stat3 and correlated with Src transformation. These findings demonstrate that Src can activate STAT signaling pathways and raise the possibility that Stat3 contributes to oncogenesis by Src.
Content may be subject to copyright.
Journal of the American Society of Nephrology 2543
A Novel Variant of the p-Subunit of the Amiloride-Sensitive
Sodium Channel in African Americans1
Yan Ru Su,2Mark P. Rutkowski,2 Charles A. Klanke, Xiaomin Wu, Yong Cul, Paymund Y.K. Pun,
Vicki Carter, Max Reif, and Anil G. Menon3
YR. Su, CA. Klanke. X. Wu. V. Cui. AG. Menon,
Department of Molecular Genetics, Biochemistry and
Microbiology. University of Cincinnati Medical Center,
Cincinnati. OH
M.P. Rutkowski, V. Carter, M. Reif, Department of
Internal Medicine, Division of Nephrology and Hyper-
tension, University of Cincinnati Medical Center. Cm-
cinnati, OH
R.Y.K. Pun, Department of Molecular and Cellular
Physiology. University of Cincinnati Medical Center.
Cincinnati. OH
(J. Am. Soc. Nephrol. 1996; 7:2543-2549)
The amiloride-sensitive sodium channel is responsible
for the rate-limiting step of sodium reabsorption in the
distal renal tubule, and thus may play a key role in the
maintenance of sodium balance and blood pres-
sure. In this study, a genetic variant that results in a
change ofthreonine to methionine at amino acid 594
(T594 M) in the carboxy-terminus of the p-subunit of
the amiloride-sensitive sodium channel has been
identified. This variant was present in 6.1 % of African-
American subjects (N =231) but was not seen in
Caucasians (N =192). Whole cell voltage clamp of
B-lymphocytes from individuals with the T594 M van-
ant showed similar basal membrane slope conduc-
tance, compared with the wild-type but increased
response to cAMP analog.
Key Words: Blacks. blood pressure, ion channels. patch
clamp techniques. sodium balance
The regulation of sodium balance in mammals is a
process that involves a number of sodium trans-
porters. many of which are expressed in the kidney.
Exact sodium balance is ultimately determined by the
distal nephron. Because the amiboride-sensitive so-
dium channel is a key regulator of salt reabsorption in
the nephron. we booked for mutations in the gene
encoding the 3-subunit of this channel in African
1Received June 4, 1996. Accepted July 23. 1996.
2Both authors contributed equally to this work.
3Correspondence to Dr. AG. Menon, Department of Molecular Genetics, Bio-
chemistry and Microbiology University of Cincinnati Medical Center, 231 Be-
thesda Avenue, Cincinnati OH 45267O524.
104&6673/07 12-2543103.00/0
Journal of the American Society of Nephrology
Copyright © 1996 by the American Society of Nephrology
Americans-a group known to have a higher preva-
lence of both hypertension and salt-sensitivity when
compared with Caucasians ( 1-5). The channel Is corn-
posed of at beast three subunits: a, 3, and y (6.7). The
human genes encoding all three subunits have been
identified and cloned (8-1 1). Mutations In the car-
boxy-terminus of the f3 and y subunits of this channel
have been proposed to be the underlying cause of
Llddbe’s syndrome. a rare autosomab dominant form of
hypertension ( 1 2, 1 3). The f3-subunit of the amiboride-
sensitive sodium channel has been shown to be ex-
pressed in the renal collecting duct (14) and in B-
lymphocytes ( 15. 16). and the electrophysioboglcal
characteristics of the channel in these two tissues
have been shown to be indistinguishable ( 1 7). Whole
cell patch clamp studies in B-lymphocytes from mdi-
viduals with Liddle’s syndrome ( 1 8) and expression of
Liddle’s syndrome mutant channels in Xenopus oo-
cytes ( 1 9) have shown increased channel activity.
More recently, this increased sodium conductance
has been correlated with an increased number of
channels on the oocyte membrane (20).
We report a novel variant that results in a substitu-
tion ofrnethionine for threonine at Position 594. In the
carboxy-terminus of the 3-subunit. This variant was
detected in African Americans but not in Caucasians.
Whole cell voltage clamping in B-lymphocytes show
that the variant was associated with enhanced so-
dium conductance in response to a cAMP analog.
Subjects were recruited from the Hypertension Clinics of
the University of Cincinnati and the Veterans Administration
Hospital, as well as from the community at large. The study
was approved by the Institutional Review Board of the Uni-
versity of Cincinnati.
African Americans
All subjects underwent a brief clinical evaluation and
provided a blood sample for measurement ofplasma electro-
lytes and DNA isolation. Subjects were seated for 5 mm. after
which blood pressure recordings were done in triplicate by
using a mercury manometer with the appropriate cuff size
based on the upper mid-arm circumference. Korotkoff Phase
V was used to define the diastolic pressure.
Treated hypertensive subjects were included if they re-
ported that the onset ofhypertenslon was before the age of 60
and that they had been under continuous treatment with
antihypertensive medication for the previous 6 months. In
hypertensive subjects not on antihypertensive treatment,
average diastolic blood pressure was confirmed to be 90
mm Hg on a second visit a week later. All hypertensive
subjects met the following criteria: no reported history of
secondary hypertension: serum potassium level 3.5 mEq/L
(unless they were being treated with thiazide or loop diuret-
Amiloride-Sensitive Sodium Channel Variant
254.4 Volume 7 -Number 12 -1996
Ics); serum creatinine concentration 1 .6 mg/dL for men
and s 1 .4 mg/dL for women; alcohol intake 1 .5 ounces of
ethanol per day: and no use of steroids or estrogen before the
diagnosis of hypertension. In subjects with non-insulin-
dependent diabetes mellitus, the diagnosis of hypertension
had to precede the diagnosis of diabetes mellitus by at beast
5 yr.
Normotensive subjects were included if systolic pressure
was <150 and diastolic pressure was <90 mm Hg. based on
the average of three readings on a single visit while not being
treated with antlhypertensive medications.
To determine allele frequency of the T594 M variant In
Caucasians. two populations were combined: ( 1) 1 20 unre-
lated grandparents from the Centre d’Etude du Polymor-
phisme Humain (CEPH) pedigree for whom hypertension
status is unknown (2 1 ), and (2) 72 unrelated Caucasians
from the Cincinnati area (30 normotensive, 32 hypertensIve.
and ten of indeterminate blood pressure status).
DNA Analysis
DNA was extracted from peripheral blood by using Pure-
gene DNA isolation kit (Gentra Systems Inc. Minneapolis.
MN). Single-strand conformatlonal polymorphism analyses
were performed by amplifying DNA fragments of the final
exon of human amlborlde-sensitive sodium channel 3-sub-
unit (-ENaC) from genomic DNA. Oligonucleotide primers
ENaC 1746 and f3ENaC 1940 were used in polymerase chain
reactions. as previously described ( 1 2). Products were frac-
tionated on 0.6X MDE (Mutation Detection Enhancement)
gels (AT Biochem. Malvern. PA) and 0.8X TBE (Tris Borate
EDTA) as electrophoresis buffer at room temperature for 12
to 1 4 h. After autoradiography. the variant band was cut
from the gel. and the DNA was eluted and reamplified before
being sequenced with an ABI automated DNA sequencer
(ABI. Foster City, CA). In all cases, both strands of DNA were
Aldosterone Profiling
Eight subjects with the T594 M variant. of which four were
hypertensive. were randomly chosen for aldosterone profiling
and ebectrophysiobogical evaluations. Eight African-Amen-
can subjects without the T594 M variant (ofwhom three were
hypertensive) were chosen as control subjects such that age,
sex. and body mass index were matched between the two
groups. This aldosterone profiling protocol has been used to
classify Individuals in Liddbe’s syndrome kindreds (22). All
subjects collected urine overnight. At approximately 8 am.
the next day. blood was collected after the subject sat upright
for 30 mm. All subjects continued to follow their usual diet
and medication treatment. Serum and urine aldosterone
levels were measured by RIA (Coat-A-Count: Diagnostic
Product Corporation. Los Angeles. CA). Normal ranges for the
upright position are 5 to 30 ng/dL for serum aldosterone and
2 to 14 g/24 h for urine abdosterone. Although no normal
range has been established for overnight 1 2-h collections,
12-h aldosterone excretion rates have been shown to come-
late with 24-h collection excretion rate in adults (23). Plasma
renin samples were determined by RIA (New England Nu-
clear. Boston, MA). The normal range for upright plasma
renin with this assay Is 0.3 to 3.0 ng/mL per h.
Electrophysiological Studies
The electrophyslologlcal measurements on lymphocytes
were conducted In a single-blind manner (presence or ab-
sence of the variant was not revealed to experimenter). The
bath medium was RPMI 1640 contaIning low Ca2 (0.54 mM)
supplemented with 10 mM N-hydroxyethylpiperazmne-N’-2-
ethanesulfonic acid to buffer the pH. The perforated-patch
method was used (24,25). The pipette was tip-filled with
recording medium containing (in mM): KC1, 70; K2S04. 30;
NaC1, 12; EGTA, 0.5: MgCl2. 1: HEPES, 20; glucose, 10 (pH
was adjusted to 7.2 and osmolarity to 300 mosmol). The
pipette was then back-filled with nystatin-containing record-
ing solution (200 to 300 tg/mL) prepared from a fresh stock
consisting of 50 mg/mL of nystatin sonicated in dimethyb
sulfoxide. Resistances of the pipettes were between 12 to 15
The slope conductance was measured from the linear
portion of the ramp current elicited by a voltage ramp from
-150 to +50 mV over the duration of 1 s, using the software
pCbamp (Version 5.5. Axon Instruments, Foster City, CA). At
beast four ramps at 0.2 Hz were averaged for each measure-
ment. After the control ramps were obtained (two to three
measurements), 300 j.tM 8-CPT-cAMP (8-CPT 8-(4-chboro-
phenylthio) adenosine 3.5-cyclic monophosphate). a mem-
brane-permeabbe analog of cAMP, was supemfused onto the
cell from a blunt-tipped pipette (5 to 10 .tm) placed nearby,
and two to three more measurements were performed. Cur-
rent records were filtered at 5 kHz. digitized, and stored for
Statistical Analysis
Results are expressed as mean ±SD or 95% confidence
Intervals. Plasma renin bevels, abdosterone bevels, sodium
and potassium excretion rates, and ratio of urine aldosterone
to potassium were log-transformed before analysis of van-
ance. To correlate the aldostemone profile results with the
electrophysioboglc data. the ratio of the 8-CPT-cAMP-stimu-
lated to basal conductance was averaged for each subject
and used as the dependent variable in an analysis of variance
(Statistical Analysis System; SAS Institute, Cary, NC).
Identification of a Missense Mutation at Codon
594 of the Amiloride-Sensitive Sodium Channel
f3 Subunit
Single-strand conformationab polymorphism analy-
sis of the 3’ end of the gene showed a variant band in
some individuals of African-American descent. Elu-
tion of this band and subsequent sequence analysis
revealed a single nucleotide substitution (C-T) In the
coding strand at amino acid position 594. Sequencing
the opposite strand confirmed the initial observation.
The normal allele was sequenced and found to be
identical to that reported by McDonald et at. (10).
These results are shown in Figure 1 .All individuals
with this variant were heterozygous at this locus.
The frequencies of the T594 M variant are shown in
Table 1 .The clinical characteristics of the African
Americans with and without the T594 M variant are
shown in Table 2. There were no statistically signifi-
cant differences between African Americans with and
without the T594 M.
a 2 12.2, p =0.002 for the entire table.
bf.0.12. P=0.72 versus hypertensive subjects.
C 2 =]2.0. P=0.0005 versus African-American subjects (first two rows
Su et al
Journal of the American Society of Nephrology 2545
Figure 1.Sequence comparison of both strands of DNA from
wild-type and variant. Panel A shows a comparison of
wild-type sequence with sequence of sense strand. The C-IT
transition is highlighted with an asterisk. Panel B shows
comparison of wild-type sequence with anti-sense strand.
The complementary G-A transition is highlighted with an
Amiloride-Sensitive Sodium Conductance in
Lymphocytes With and Without the T594 M
Membrane slope conductance of isolated B-bympho-
cytes from individuals without the T594 M variant
(wild-type) and B-lymphocytes harboring the T594 M
variant were measured. Slope conductances before
and in the presence of 8-CPT-cAMP from the two
groups were then compared. The basal slope conduc-
tances for the two groups were similar: 0.43 ± 0.24 nS
(N =15) forwild-type cells and 0.45 ±0.21 nS(N =12)
for T594 M variant cells (P =0.79). In the presence of
8-CVT-cAMP, slope conductance of responsive cells
TABLE 1.T594M variant in populations of unrelated
Group NT594M/
NTested Percentagea
African-American Hypertensive
African-American Normotensive
Caucasian Subjects
was increased to 1 .20 ±0.72 nS for the wild-type cells
(paired t test, P<0.05) and 1 .99 ±1 .22 nS for the
variant cells (paired t test, P<0.05). One-way analysis
of variance shows that the 8-CPT-cAMP-stimulated
slope conductance Increase In the T594 M variant is
significantly higher than that for the wild-type (P <
0.05). Representative traces of the recordings ob-
tamed are shown in Figure 2. Because the amiboride-
sensitive sodium channel Is most active at very nega-
tive membrane potentials (more negative than -120
rnV), the presence of channel activities obscured the
measurements of the slope conductance at the nega-
tive potentials (see Figure 2). Thus, the slope conduc-
tance that we measured during 8CPT-cAMP applica-
tion is likely to be an underestimation of the actual
changes that had occurred.
The change in slope conductance in response to
8-CPT-cAMP measured for the variant could result
from a higher number of amiboride-sensitive sodium
channels (also an increase in probability of channel
opening. unitary conductance. etc.) present in the
variant lymphocytes or from a change in responsive-
ness of the channel to 8-CPT-cAMP. To compare the
responsiveness of the amiboride-sensitive sodium
channel with 8-CPT-cAMP in the wild-type and the
variant, and to rule out the possibility that the re-
sponse is rebated to the basal conductance, we exam-
med the ratio of the 8-CPT-cAMP response to basal
slope conductance in the same cell. The response ratio
was 2.97 ±1.13 (N =15) for the wild-type. This ratio
is in good agreement with the ratio we derived (3.12)
from the data reported for normal B-lymphoid cells by
Oh et at.. ( 15). For the variant, the response ratio was
4.72 ±1.94 (N =12), which is significantly higher
when compared with the wild type (P =0.007). These
results indicate that the T594 M variant is more
responsive to 8-CPT-cAMP.
When amiloride (2 jIM) was coapplied with 8CPT-
cAMP, the slope conductance were 0.36 ± 0.24 nS
(N =10: control subjects without arniboride 0.39 ±
0.23 nS) and 0.57 ±0.30 nS (N =7; control subjects
without amiboride, 0.55 ± 0.26) for wild-type and T594
M variant, respectively (Figure 2). The result showed
that the enhancement in slope conductance by 8-CPT-
cAMP occurs via an arniboride-sensitive conductance.
Amiloride-Sensitive Sodium Channel Variant
2546 Volume 7 -Number 12 -1996
TABLE 2. Clinical characteristics of African-American subjects#{176}
Group Genotype N (mit) Age (yr) Age Dx
(yr) BMI
(kg/m2) SBP
(mm Hg) DBP
(mm Hg) Rx
Hypertensive Subjects Wild-type 1 19 (47/72) 52 ±11 39 ±12 32.6 ±7.9 144 ±22 92 ±14 87
Hypertensive Subjects T594M 7 (5/2) 62 ±15 44 ±16 28.4 ±3.8 149 ±18 91 ±6 87
Normotensive Subjects Wild-type 98 (30/68) 57 ±17 n/a 28.1 ±6.9 124 ±14 77 ±7 0
Normotensive Subjects T594M 7 (3/4) 55 ±14 n/a 27.2 ±4.2 125 ±10 77 ±8 0
0Values shown are mean SD. N. number of subjects; m/f, Nmale/Nfemaie; Age Dx, age of diagnosis of hypertension; SBP. systolic blood pressure;
DBP, diastolic blood pressure; Rx, subjects on antihypertensive medications; BMI. body mass index.
Aldosterone Profiles of Subjects With and
Without T594 M
The aldosterone profiles of the 16 subjects whose
lymphocytes were studied electrophyslobogically are
shown In Table 3. Although the serum aldosterone
and urine abdosterone bevels were statistically higher
In the subjects with the T594 M variant. there was no
statistical difference when the urine aldosterone bevel
was normalized for potassium excretion. The levels of
circulating aldosterone in individuals with the T594 M
variant were of particular Interest because earlier
studies in mammalian nephrons show that aldoste-
rone increases the Na+ conductance in the apical
membrane through the activation of amiboride-sensi-
tive sodium channel (26,27). To assess whether the
higher abdosterone bevels observed in Individuals with
the T594 M variant could account for the higher
8-CPT-cAMP-stimubated slope conductance in the
lymphocytes from these individuals. an analysis of
variance was performed using the ratio of 8-CPT-
cAMP-stimulated- to-basal slope conductance as the
dependent variable. The covariates urine sodium ex-
cretlon, urine potassium excretion, and abdosterone
excretion did not abolish the relationship between
presence of the variant and higher 8-CPT-cAMP-stlm-
ubated slope conductance (P =0.00 1).
We have Identified a missense mutation T594 M in
the carboxyb terminus of the 13-subunit of the amibo-
ride-sensitive sodium channel that is present in nor-
motensive and hypertensive African Americans (6.1%)
but not in Caucasians. Ebectrophysbobogicab studies of
the amiboride-sensitive sodium conductance In bym-
phocytes revealed that this variant is associated with
an enhanced 8-CPT-cAMP-stlmulated response when
compared with wild-type (4.72- versus 2.97-fold. re-
There Is evidence that mutations in the carboxy-
terminus of the 3 subunit of the amiloride-sensitive
sodium channel can bead to abnormal channel behav-
ior, causing sodium retention and hypertension. Mu-
tatlons causing a premature truncation of the last 43
to 75 amino acids of the carboxy-termmnus of the
amiloride-sensitive sodium channel a-subunIt have
been Identified in Liddle’s syndrome. an autosomal
dominant form of hypertension (12). The T594 M
variant that we have identified is located within this
region (47 amino acids from the carboxy-termmnus).
Our electrophysioboglc and clinical data for the T594
M variant differ from that reported for Liddbe’s syn-
drome. The amiboride-sensitive sodium channels of
B-bymphoid cells from Liddle’s patients did not re-
spond to 8-CPT-cAMP ( 1 8), whereas the response to
8-CPT-cAMP in the T594 M variant was enhanced
when compared with the wild-type. This lack of re-
sponse in Liddle’s syndrome has been attributed to
increased inward baseline sodium current. Recently,
it has been reported that Liddle’s syndrome trunca-
tion mutations in the carboxy terminus of the a-sub-
unit result in an increased number of channels ex-
pressed in Xenopus laevis oocytes ( 1 9). Analysis of the
carboxy-terminus revealed a conserved eight-amino-
acid motif(PPPXYXXL) that when mutated, resulted in
an increased number of sodium channels expressed
on the cell surface (20). Although we cannot rube out
whether the enhanced responses induced by 8-CPT
cAMP in the T594 M variant result from an increase in
the channel density on the cell surface, the increase in
response ratio suggests that the enhanced slope con-
ductance is likely a result of a change in an Intrinsic
property of the channel. Thus It Is possible that the
physiological robe played by the T594 M variant (which
is not contained within the PPPXYXXL motif) may be
different from variants observed in Liddle’s syndrome.
The back of suppression of aldosterone in individuals
with the T594 M variant and Its presence In normo-
tensive individuals (five of seven of whom were over
the age of 50) also contrasts with Liddbe’s syndrome
patients, in whom abdosterone secretion is suppressed
secondary to sodium retention and in whom hyperten-
sion usually develops during the second decade of life
Other investigators have suggested that cAMP can
either activate or inhibit the activity of the amiboride-
sensitive sodium channel. Bubien et at. .have shown
in lymphocytes that cAMP usually activates the amilo-
ride-sensitive sodium conductance but inhibits chan-
neb activity after exposure to pertussis toxin (28).
Stutts et at. .reported that coexpressing the cystic
fibrosis transmembrane regulator with the rat renal
amlboride-sensitive sodium channel suppressed
cAMP-stimulated channel activity (29). Our studies
indicate that lymphocytes expressing the T594 variant
-1 50 mV
Variant 1
-1 50 mV
Variant 2
100 msec
Variant 3
-1 50 mV
-1 50 mV
Su et al
Journal of the American Society of Nephrology 2547
Figure 2. Current-Voltage (l-V) relation measured in lymphocytes from Individuals withoutthe variant (left panel, wild-type) and
from individuals harboring the variant (right panel) under perforated-patch recording. I-V relation was elicited and the slope
conductance obtained as described in the method section. Trace A in both panels shows the I-V relation under control conditions.
Trace B In each panel shows the I-V relation in the presence of 8-CPT-cAMP. Trace C in both panels shows the I-V relation in the
presence of both of 8-CPT-cAMP and amilonide (2 1.tM). Variants are representative fracings from three different individuals.
sodium channel not only show an enhanced response
to cAMP but also show a greater susceptibility to
opening at negative potentials on some occasions.
possibly because of the boss of a suppressive effect.
The threonine residue at position 594 in the a-subunit
may be Involved in the regulation of channel activity,
as It Is a potential target for phosphorybation by
protein kinase C (PKC) (10). It has been shown, for
example. that activation of PKC by addition of phorbol
1 2, 1 3-dibutyrate or mezerein abolished amiboride-
sensitive sodium channel activity (30). The substitu-
tion of methionine for threonine at this site may
therefore cause the channel to become resistant to the
negative-regulatory effect of PKC, and could explain
the enhanced response to 8-CPT-cAMP seen in the
T594 variant. It is possible that the mechanism of
regulation of channel activity involves at least two
distinct sites-a positive modulatory site that in-
creases channel activity and a negative modulatory
site that decreases channel activity. The sites may be
on the sodium channel itself or on its associated
regulatory complex. Regulatory sites other than the
cAMP and PKC sites, such as G protein binding sites,
may also exist (28).
An important and yet unresolved question Is
whether this variant, apparently specific to African
Americans. plays a role In the higher prevalence of
salt-sensitive hypertension seen in this population.
Although there was no significant difference In the
prevalence of the T594 M variant between the hyper-
Amiloride-Sensitive Sodium Channel Variant
2548 Volume 7 -Number 12 -1996
TABLE 3. Aldosterone profile of a subset of African-American subjects with and without T594M vaniant#{176}
Parameter Wild-Type (N =8) T594M Variant (N =8)
Serum Potassium (mEq/L) 4.2 (3.9 to 4.6) 4.5 (4.3 to 4.7)
Plasma Renin Activity (ng/mL per h) 0.45 (0. 19 to 1.04) 0.37 (0. 17 to 0.78)
Serum Aldosterone (ng/dL) 6.9 (4.4 to 10.8) 13.3 (9.9 to 17.9)b
Urine Sodium (mEq/ 12 h) 70 (55 to 89) 62 (40 to 62)
Urine Potassium (mEq/12 h) 21 (14 to 32) 27 (17 to 41)
Urine Aldosterone (g/12 h) 2.0 (1.2 to 3.2) 4.4 (2.8 to o.7)b
Aldosterone:Potassium Ratioc 94 ( to 135) 172 (92 to 325)
aincludes four hypertensive and four normotensive subjects in the T594M group. and three hypertensive and five normotensive subjects in the
wild-type group. All values reported except potassium are the anti-log of the mean log-transformed value (95% confidence intervals).
bp 0.03 by analysis of variance; all others are not significantly different.
CNanograms of urine aldosterone per milliequivalent of urine potassium.
tensive and normotensive populations that we stud-
led, this does not preclude it from being one of the
contributing factors to the trait. The phenotypic con-
tributlon of the T594 M variant may not be evident in
normotensive subjects who harbor the variant with-
out the additive effects ofother genetic or environmen-
tab factors.
It Is Important to point out some of the limitations in
the interpretation of our observations. First, our elec-
trophysiobogical studies were whole cell patch clamp-
Ing measurements and not single channel recordings.
Second, although it has been shown by reverse tran-
scrlption- pobymerase chain reaction that mRNA en-
coding the 3- subunit is present in lymphocytes from
human (15) and rat (16), the alpha subunit of this
channel is probably a different isoform both in rat and
human lymphocytes from the alpha subunit ex-
pressed in the renal collecting duct (15, 16).
Nonetheless, It has been shown that amiboride-
sensitive sodium channel activity is increased in lym-
phocytes from individuals with Liddle’s syndrome
(similar to what has been observed in the oocyte
expression system of Liddle’s mutations). Therefore,
although it is possible that the difference in whole cell
ebectrophyslobogicab activity conferred by the n-sub-
unit variant T594 M in B-lymphocytes would also be
present in the sodium channel expressed in the renal
collecting tubule. this has not been proven in our
study. If this variant does predispose to salt-sensitive
hypertension, it would have to alter the function of the
amilorlde-sensltive sodium channel in the renal col-
becting duct.
Whether the T594 M variant confers any selective
advantage to individuals who harbor the variant gene
remains unknown. It is possible that the increased
ability to reabsorb sodium conferred by the variant
channel may be an advantage In certain conditions of
salt deprivation, or, alternatively. increased protection
against volume depletion in conditions of water scar-
The dIscovery of the T594 M genetic variant in the
13-subunit of the amiloride-sensitive sodium channel,
a key regulator of renal sodium handling, and the
altered electrophysiobogicab properties associated with
it may be one of the first steps in establishing the
amiboride-sensitive sodium channel as a genetic com-
ponent of essential hypertension in African Amen-
This work was supported in part by grants from the National Insti-
tutes of Health Program of Excellence and Markey Foundation to
A.G.M. .from Dialysis Centers Inc. to M.P.R and MR. and a Cardlo-
vascular Center Research Grant from the University ofClnclnnati. We
thank Drs. Peter Gartslde. Kenneth Blumenthal. John Cupoletti,
Jerry Lingrel. Robert Luke. and Gary Shull for discussions and
helpful comments on the manuscilpt. and acknowledge the excellent
technical assistance ofTerri Lewis and Kristen Braig.
1.Cornoni-Huntley J. LaCrolz AZ, Havlik RJ: Race and sex
differentials in the impact of hypertension in the United
States: The National Health and Nutrition Examination
Survey I epidemlological follow-up study. Arch Intern
Med 1989:149:780-788.
2. Weinberger MY, Miller JZ, Luft FC, Grim CE, Fineberg
NS: Definitions and characteristics of sodium sensitivity
and blood pressure resistance. Hypertension 1986:
8ISuppl 21:127-134.
3. Falkner B, Kushner H: Effect of chronic sodium loading
on cardiovascular response in young blacks and whites.
Hypertension 1990; 15:36-43.
4. Harshfield GA, Alpert BS, Pulliam DA, Willey ES, Somes
GW, Stapleton FB: Sodium excretion and racial differ-
ences in ambulatory blood pressure patterns. Hyperten-
slon 1991:18:813-818.
5. Luft FC, Grim CE, Fineberg NS, Wemnberger MH: Effects
of volume expansion and contractions In normotensive
whites, blacks, and subjects ofdifferent ages. Circulation
1 979:59:643- 650.
6. Canessa CM, Horisberger JD. Rossier BC: Epithelial
sodium channel related to proteins involved in neurode-
generation. Nature (Lond) 1993:361:467-470.
7. Canessa CM, Schild L, Buell G, et at.: Amiboride-
sensitive Na  channel is made of three homologous
subunits. Nature (Lond) 1994;367:463-467.
8. McDonald FJ, Snyder PM, McCray PB Jr. Welsh MJ:
Cloning, expression. and tissue distribution of a human
amilonide-sensitive Na channel. Am J Physiol 1994;
9. Voilley N, Lingueglia E. Champigny G, et al.: The lung
amilonide-sensitive Na channel: Biophysical properties.
pharmacology. ontogenesis. and molecular cloning. Proc
Natl Acad Sci USA 1994:91:247-25 1.
1 0. McDonald FJ, Price MP, Snyder PM. Welsh MJ: Cloning
and expression of the beta- and gamma-subunits of the
human sodium channel. Am J Physlol 1 995;268:C 1157-
Cl 163.
Sut dl
Journal of the American Society of Nephrology 2549
1 1 .Voilley N, Bassilana F, Mignon C, et at.: Cloning. chro-
mosomal localization, and physical linkage of the 3 and
‘/ subunits (SCNN1B and SCNN1G) ofthe human amilo-
ride-sensitive sodium channel. Genomics 1 995; 28:560 -
12. Shimkets RA. Warnock DG, Bositis MC, et aL: Liddle’s
Syndrome: Heritable human hypertension caused by
mutations in the j3 subunit of the sodium channel. Cell
13. Hansson JH, Nelson-Williams C, Suzuki H, et al.: Hy-
pertension caused by a truncated sodium channel y
subunit: Genetic heterogeneity of Liddle syndrome. Nat
Genet 1995;1 1:76-82.
14. Duc C, Farman N, Canessa CM, Bonvalet JP, Rossier
BC: Cell-specific expression of sodium channel a, 3. and
.y subunits in abdosterone responsive epithelia from the
rat: Localization by in situ hybridization and immunocy-
tochemistry. J Cell Biol 1994:127:1907-1921.
15. Oh Y. Bubien JK, Warnock DG: RT-PCR analysis of
amiloride-sensitive sodium channel (ASSC) subunit
mRNAs from human B cells lAbstractl. J Am Soc Neph
16. Bradford AL, Ismailov II, Achard J, Warnock DG, Bu-
bien JK, Benos DJ: Immunopunificatlon and functional
reconstitution of a Na  channel complex from rat lym-
phocytes. Am J Physiol 1995;269:C601-C61 1.
1 7. Bubien JK, Warnock DG: Amibonide-sensitive sodium
conductance in human B lymphold cells. Am J Physlol
1993:265:C1 175-Cl 183.
18. Warnock DG, Bubien JK: Liddle’s syndrome: Clinical
and cellular abnormalities. Hosp Pract 1994;29:95-105.
19. Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton
pp. Rossier BC: A mutation In the sodium channel
causing Liddle disease increases channel activity In the
xenopus laevis oocyte expression system. Proc NatI Acad
Sd USA 1995:92:5699-5703.
20. Snyder P. Price MP, McDonald FJ, et al.: Mechanism by
which Liddle’s syndrome mutations increase activity of a
human Na channel. Cell 1995;83:969-978.
2 1 .Dausset J, Cann H, Cohen D, Lathrop GM, Labouel JM,
White RI: Centre d’Etude du Polymorphisme Humain
(CEPH): Collaborative genetic mapping of the human
genome. Genomics 1990:6:575-577.
22. Botero-Velez M, Curtis J, Warnock DG: Brief report:
Liddle’s syndrome revisited-A disorder of sodium reab-
sorption in the distal tubule. N Engl J Med 1994:330:
23. Pratt JH, Miller JZ, Fineberg NS, Parkinson CA: Aldo-
sterone excretion rates in children and adults during
sleep. Hypertension 1986:8:154-158.
24. Horn R, Marty A: Muscaninic activation of Ionic currents
measured by a new whole-cell recording method. J Gen
Physlol 1988;92: 145-159.
25. Pun RYK, Kleeneand SJ, Gesteland RC: Guanine nude-
otides modulate steady-state Inactivation of voltage-
gated sodium channels In frog olfactory receptor neu-
rons. J Membr Blob 1994; 142: 103-1 1 1.
26. ReifMC, Troutman SL, SchaferJA: Sodium transport by
rat cortical collecting tubule: Effect of vasopressin and
desoxycorticosterone. J ClIn Invest 1986:77: 129 1-1298.
27. Schafer JA, Hawk CT: Regulation of Na +channels In the
cortical collecting ducting duct by AVP and minerabocor-
ticoids. Kidney mt 1992:41:255-268.
28. Bubien JK, Jope RS, Warnock DG: G-protelns modulate
amibonide-sensitive sodium channels. J Blol Chem 1994;
29. Stutts MJ, Canessa CM, Olsen JC et al.: CFTR as a
cAMP-dependent regulator of sodium channels. Science
30. Yanase M, Handler JS: Activations of protein kinase C
inhibit sodium transport In A6 epithella. Am J Physlol
1986:250:C5 17-C522.
... c-Src tyrosine kinase can constitutively activate STAT3, which increases the possibility of the STAT signaling pathway regulating tumor-related gene expression. 155 Epidermal growth factor receptor can directly activate STAT1, STAT3, and STAT5, furthermore, STAT5 can be directly activated by the platelet-derived growth factor receptor. 99,156,157 Positive regulation of JAK/STAT signaling In addition to the main components of the JAK/STAT signaling pathway, many related proteins play indispensable roles in STATdependent transcription and JAK-STAT interactions with other signaling pathways. ...
Full-text available
The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway was discovered more than a quarter-century ago. As a fulcrum of many vital cellular processes, the JAK/STAT pathway constitutes a rapid membrane-to-nucleus signaling module and induces the expression of various critical mediators of cancer and inflammation. Growing evidence suggests that dysregulation of the JAK/STAT pathway is associated with various cancers and autoimmune diseases. In this review, we discuss the current knowledge about the composition, activation, and regulation of the JAK/STAT pathway. Moreover, we highlight the role of the JAK/STAT pathway and its inhibitors in various diseases.
... The docking model between the STAT3 protein and STA-21, identified by structure-based virtual screening, has predicted that STA-21 will bind to the Tyr-705 site of STAT3 and form a number of hydrogen bonds between Tyr-705 and nearby residues [7]. The activation of the STAT3 Tyr-705 site induces dimerization of STAT3 in the cytosol, and the dimer is translocated into the nucleus to act as a transcription factor [30][31][32][33][34]. Therefore, the inhibition of Tyr-705 could be a target in the treatment of disease containing the STAT3 signaling pathway. ...
Full-text available
The activation of signal transducer and activator of transcription 3 (STAT3), as well as up-regulation of cytokines and growth factors to promote STAT3 activation, have been found in the epidermis of psoriatic lesions. Recently, a series of synthetic compounds possessing the Michael acceptor have been reported as STAT3 inhibitors by covalently binding to cysteine of STAT3. We synthesized a Michael acceptor analog, SKSI-0412, and confirmed the binding affinity between STAT3 and SKSI-0412. We hypothesized that the SKSI-0412 can inhibit interleukin (IL)-17A-induced inflammation in keratinocytes. The introduction of IL-17A increased the phosphorylation of STAT3 in keratinocytes, whereas the inactivation of STAT3 by SKSI-0412 reduced IL-17A-induced STAT3 phosphorylation and IκBζ expression. In addition, human β defensin-2 and S100A7, which are regulated by IκBζ, were significantly decreased with SKSI-0412 administration. We also confirmed that SKSI-0412 regulates cell proliferation, which is the major phenotype of psoriasis. Based on these results, we suggest targeting STAT3 with SKSI-0412 as a novel therapeutic strategy to regulate IL-17A-induced psoriatic inflammation in keratinocytes.
... Lupeol suppresses constitutive activation of c-Src. Signal transducer and activator of transcription signalling 3 has also been reported to be activated by soluble tyrosine kinases of the Src kinase families (Yu et al, 1995;Subramaniam et al, 2013). Hence, we determined the effect of lupeol on constitutive activation of Src kinase in HepG2 cells. ...
... We also showed that SRC V177M upregulates STAT3 at mRNA level. Although several studies have reported an increase in STAT3 transcriptional activity by SRC phosphorylation leading to gene expression of STAT3 target genes [46][47][48], little is known about the transcriptional regulation of STAT3 itself, potentially involving SRC protein. A possible explanation approach may include the tumor suppressor protein p53, downregulated in our study by the SRC V177M variant. ...
Full-text available
Colorectal cancer (CRC) shows one of the largest proportions of familial cases among different malignancies, but only 5–10% of all CRC cases are linked to mutations in established predisposition genes. Thus, familial CRC constitutes a promising target for the identification of novel, high- to moderate-penetrance germline variants underlying cancer susceptibility by next generation sequencing. In this study, we performed whole genome sequencing on three members of a family with CRC aggregation. Subsequent integrative in silico analysis using our in-house developed variant prioritization pipeline resulted in the identification of a novel germline missense variant in the SRC gene (V177M), a proto-oncogene highly upregulated in CRC. Functional validation experiments in HT-29 cells showed that introduction of SRCV177M resulted in increased cell proliferation and enhanced protein expression of phospho-SRC (Y419), a potential marker for SRC activity. Upregulation of paxillin, β-Catenin, and STAT3 mRNA levels, increased levels of phospho-ERK, CREB, and CCND1 proteins and downregulation of the tumor suppressor p53 further proposed the activation of several pathways due to the SRCV177M variant. The findings of our pedigree-based study contribute to the exploration of the genetic background of familial CRC and bring insights into the molecular basis of upregulated SRC activity and downstream pathways in colorectal carcinogenesis.
... In 1995, Yu et al. (1995) discovered for the first time that the abnormal activation of STAT3 is related to the occurrence and FIGURE 7 | Stattic combined with VP-16 promote MV4-11 apoptosis. MV4-11 cells were treated with DMSO (control), Stattic (2.5 µM), and VP-16 (4 µg/ml) alone or in combination for 4.5 h. ...
Full-text available
The FMS-like tyrosine kinase 3 (FLT3)- internal tandem duplication (ITD) mutation can be found in approximately 25% of all acute myeloid leukemia (AML) cases and is associated with a poor prognosis. The main treatment for FLT3-ITD-positive AML patients includes genotoxic therapy and FLT3 inhibitors, which are rarely curative. Inhibiting STAT3 activity can improve the sensitivity of solid tumor cells to radiotherapy and chemotherapy. This study aimed to explore whether Stattic (a STAT3 inhibitor) affects FLT3-ITD AML cells and the underlying mechanism. Stattic can inhibit the proliferation, promote apoptosis, arrest cell cycle at G0/G1, and suppress DNA damage repair in MV4-11cells. During the process, through mRNA sequencing, we found that DNA damage repair-related mRNA are also altered during the process. In summary, the mechanism by which Stattic induces apoptosis in MV4-11cells may involve blocking DNA damage repair machineries.
... We also showed that SRC V177M upregulates STAT3 at mRNA level. Although several studies have reported an increase in STAT3 transcriptional activity by SRC phosphorylation leading to gene expression of STAT3 target genes [46][47][48], little is known about the transcriptional regulation of STAT3 itself, potentially involving SRC protein. Even though the exact underlying mechanisms remain to be elucidated, our results indicate that the SRC V177M variant and thus active pSRC Y419 may increase STAT3 gene expression and may contribute to CRC. ...
Full-text available
Colorectal cancer (CRC) shows one of the largest proportions of familial cases among different malignancies, but only 5-10% of all CRC cases are linked to mutations in established predisposition genes. Thus, familial CRC constitutes a promising target for the identification of novel, high- to moderate-penetrance germline variants underlying cancer susceptibility by next generation sequencing. In this study, we performed whole genome sequencing on 3 members of a family with CRC aggregation. Subsequent integrative in silico analysis using our in-house developed variant prioritization pipeline resulted in the identification of a novel germline missense variant in SRC gene (V177M), a proto-oncogene highly upregulated in CRC. Functional validation experiments in HT-29 cells showed that introduction of SRCV177M resulted in increased cell proliferation and enhanced protein expression of phospho-SRC (Y419), a potential marker for SRC activity. Upregulation of paxillin, β-Catenin and STAT3 mRNA levels, increased levels of phospho-ERK, CREB and CCND1 proteins and downregulation of the tumor suppressor p53 further proposed the activation of several pathways due to the SRCV177M variant. The findings of our pedigree-based study contribute to the exploration of the genetic background of familial CRC and bring insights into the molecular basis of upregulated SRC activity and downstream pathways in colorectal carcinogenesis.
Full-text available
Signal transducer and activator of transcription 3 (STAT3), a member of the STAT family, discovered in the cytoplasm of almost all types of mammalian cells, plays a significant role in biological functions. The duration of STAT3 activation in normal tissues is a transient event and is strictly regulated. However, in cancer tissues, STAT3 is activated in an aberrant manner and is induced by certain cytokines. The continuous activation of STAT3 regulates the expression of downstream proteins associated with the formation, progression, and metastasis of cancers. Thus, elucidating the mechanisms of STAT3 regulation and designing inhibitors targeting the STAT3 pathway are considered promising strategies for cancer treatment. This review aims to introduce the history, research advances, and prospects concerning the STAT3 pathway in cancer. We review the mechanisms of STAT3 pathway regulation and the consequent cancer hallmarks associated with tumor biology that are induced by the STAT3 pathway. Moreover, we summarize the emerging development of inhibitors that target the STAT3 pathway and novel drug delivery systems for delivering these inhibitors. The barriers against targeting the STAT3 pathway, the focus of future research on promising targets in the STAT3 pathway, and our perspective on the overall utility of STAT3 pathway inhibitors in cancer treatment are also discussed. Since the discovery of the signal transducer and activator of transcription 3 (STAT3) in 1994, the STAT3 pathway has been proven to play a pivotal role in cancer initiation, progression, and metastasis. This review summarizes the mechanisms of the STAT3 pathway regulation and the relationship between the STAT3 pathway and cancer hallmarks. The emerging development of the STAT3 pathway inhibitors, novel drug delivery systems, and perspective on the overall utility of the STAT3 pathway are also introduced.
Nicotine is an addictive ingredient of tobacco products and other noncigarette substitutes, including those being used for smoking cessation to relieve withdrawal symptoms. Earlier research, however, has associated nicotine with the risk and poorer outcome of several diseases, including cancer. Macrophages are an important component of the innate immune system and can have both pro-and anti-inflammatory functions depending upon their polarization state. Here, we investigated the effect of nicotine on macrophage polarization, growth, and invasion to understand its role in human physiology. We observed that nicotine induced M2 polarization of RAW264.7 and THP-1-derived macrophages in a dose-dependent manner. Cytokine profiling suggested a mixed M2a/d phenotype of nicotine-polarized macrophages associated with tissue repair and pro-angiogenic functions. Moreover, nicotine treatment also enhanced the growth, motility, and invasion of macrophages. Mechanistic studies revealed increased phosphorylation of STAT3 in nicotine-treated macrophages that was mediated through Src activation. Importantly, pretreatment of macrophages with either Src or STAT3 inhibitor abrogated nicotine-induced macrophage polarization, growth, and motility, suggesting a functional role of the Src-STAT3 signaling axis. Together, our findings reveal a novel role of nicotine in immunosuppression via causing M2 polarization of macrophages that could be implicated in the pathogenesis of various diseases.
Background and purpose: Cancer cachexia is one of the most common causes of death among cancer patients; no effective anti-cachectic treatment is currently available. In experimental cachectic animal models, aberrant activation of STAT3 in skeletal muscle has been found to contribute to muscle wasting. However, its clinical association, the factors regulating STAT3 activation, and the molecular mechanisms remain incompletely understood. Experimental approach: The expression of HSP90 and the activation of STAT3 were detected in muscle from the patients with cancer cachexia or the tumour-bearing cachectic mice. HSP90 inhibitors, including 17DMAG and PU-H71, were administered to cachexic mice, and cachexia parameters, such as weight loss, food intake, survival rate, body composition, serum metabolites, muscle wasting pathology, and catabolic activation, were measured and analyzed. The in vitro coculture of C2C12 myotube cells with C26 conditioned media (CM) was performed to address the pathological mechanism of catabolic muscle wasting. The roles of HSP90, STAT3, and FOXO1 in myotube atrophy were explored via overexpression or knockdown. Results: Here, we show that an enhanced interaction between activated STAT3 and HSP90, which were observed in the skeletal muscle of cancer cachexia patients, is a crucial event for the development of cachectic muscle wasting. Administration of HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 and LLC tumor-bearing cachectic mice models or the myotube atrophy of C2C12 cells induced by C26 conditional medium. Prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner in muscle cells. Conclusion and implications: Our results demonstrate that the HSP90/STAT3/FOXO1 axis plays a critical role in cachectic muscle wasting, which might serve as a potential therapeutic target for the treatment of cancer cachexia.
Full-text available
Abnormal expression of glutathione S-transferase Pi 1 (GSTP1) is associated with the progression of several tumor types. However, its role and molecular mechanism in the progression of colorectal cancer (CRC) is largely unknown. In the present study, immunohistochemistry (IHC) and quantitative-reverse transcription PCR (qRT-PCR) were used to detect the expression of GSTP1 and signal transducer and activator of transcription 3 (STAT3) in CRC tissues. Western blotting was applied to detect the expression of GSTP1 and proteins of Janus kinase (JAK)-STAT3 pathway. The interaction and co-localization of GSTP1 and STAT3 were detected by co-immunoprecipitation (CO-IP) and immunofluorescence, respectively. A positive correlation was identified between the expression of GSTP1 and STAT3 in human CRC tissues. Overexpression of GSTP1 promoted the proliferation, invasion and metastasis of CRC cells by upregulating STAT3. GSTP1 and STAT3 can directly bind to and regulate each other, and can be regulated by the upstream gene which was called F-box only protein 8 (FBX8). The present study demonstrated that GSTP1 could enhance the expression of STAT3 to promote the proliferation, invasion and metastasis of CRC cells, which provides a potential therapeutic target for clinical treatment of CRC.
Full-text available
The mechanisms by which the binding of growth hormone (GH) to its cell surface receptor elicits changes in gene transcription are largely unknown. The transcription factor Stat1/p91 has been shown to be activated by GH. Here we show that acute phase response factor or Stat3 (or an antigenically related protein), is also activated by GH. Stat3 has been implicated in the interleukin-6-dependent induction of acute phase response genes. GH promotes in 3T3-F442A fibroblasts the tyrosyl phosphorylation of a protein immunoprecipitated by antibodies to Stat3. This protein co-migrates with a tyrosyl phosphorylated protein from cells treated with leukemia inhibitory factor, a cytokine known to activate Stat3. Tyrosyl phosphorylated Stat3 is also observed in response to interferon-. Stat3 is present in GH-inducible DNA-binding complexes that bind the sis-inducible element in the c-fos promoter and the acute phase response element in the α2-macroglobulin promoter. The ability of GH to activate both Stat1 and Stat3 (i.e. increase their tyrosyl phosphorylation and ability to bind to DNA) suggests that gene regulation by GH involves multiple Stat proteins. Shared transcription factors among hormones and cytokines that activate JAK kinases provide an explanation for shared responses, while the ability of the different ligands to differentially recruit various Stat family members suggests mechanisms by which specificity in gene regulation could be achieved.
Full-text available
Interferons IFN-alpha/beta and IFN-gamma act through independent cell-surface receptors, inducing gene expression through tyrosine phosphorylation of cytoplasmic transcription factors . IFN-alpha stimulates phosphorylation and nuclear localization of the 84/91K and 113K subunits of latent ISGF3 (interferon-stimulated gene factor 3), which combine with the 48K DNA-binding subunit to bind regulatory elements of IFN-alpha-responsive genes. IFN-gamma activates p91 alone, inducing IFN-gamma-responsive genes through a distinct DNA element. Genetic complementation studies implicated the tyrosine kinase Tyk2 in IFN-alpha signalling and, more recently, the related Jak2 kinase in IFN-gamma signalling. We now present biochemical evidence for Jak-family kinase involvement in IFN signal transduction. Jak1 was activated in response to IFN-alpha and IFN-gamma; Jak2 responded exclusively to IFN-gamma. Overexpression of either Jak1 or Jak2 stimulated p91 DNA-binding activity and p91-dependent transcription. Overexpression also activated endogenous Jak kinases, suggesting that interactions between Jak kinases are required during interferon signalling.
Full-text available
Interleukin-6 (IL-6) and gamma interferon (IFN-gamma) induce a partially overlapping set of genes, including the genes for interferon regulatory factor 1 (IRF-1), intercellular adhesion molecule 1 (ICAM-1), and the acute-phase protein alpha 2-macroglobulin. We report here that the rat alpha 2-macroglobulin promoter is activated by IFN-gamma in human hepatoma (HepG2) cells and that the IFN-gamma response element maps to the same site previously defined as the acute-phase response element (APRE), which binds the IL-6-activated transcription factor APRF (acute-phase response factor). As was reported for fibroblasts, the IFN-gamma-regulated transcription factor GAF is phosphorylated at tyrosine after IFN-gamma treatment of HepG2 cells. IFN-gamma posttranslationally activates a protein which specifically binds to the alpha 2-macroglobulin APRE. This protein is shown to be identical or closely related to GAF. Although APRF and GAF are shown to represent different proteins, their binding sequence specificities are very similar. APRF and GAF bind equally well to the APRE sequences of various acute-phase protein genes as well as to the IFN-gamma response elements of the IRF-1, ICAM-1, and other IFN-gamma-inducible genes. Transient transfection analysis revealed that the IFN-gamma response elements of the IRF-1 and ICAM-1 promoters are able to confer responsiveness to both IFN-gamma and IL-6 onto a heterologous promoter. Therefore, APRF and GAF are likely to be involved in the transcriptional induction of these immediate-early genes by IL-6 and IFN-gamma, respectively. Taken together, these results demonstrate that two functionally distinct hormones, IL-6 and IFN-gamma, act through common regulatory elements to which different transcription factors sharing almost the same sequence specificity bind.
ISGF-3 is a multiprotein transcription factor that is very quickly activated in the cell cytoplasm only after attachment of interferon alpha to the cell surface. To understand the specific cytoplasmic activation of proteins that move to the nucleus and direct increased transcription of specific genes, we have purified and now report completion of the cloning of cDNA encoding the four proteins of ISGF-3. With all of the sequences available, it is clear that three of these proteins are encoded by members of a previously unrecognized gene family. We suggest that proteins encoded by this gene family serve the function of interpreting the fact that a cell surface receptor has bound its ligand so that specific signal transduction to the nucleus can occur.
We studied c-fos gene expression in rat fibroblasts by microinjection of regulatory DNA sequences, such as the serum response element (SRE) present in c-fos promotor, in order to compete directly with such sequences for binding of putative regulatory factors. We show that an additional fos intragenic regulatory element (FIRE) is located at the end of exon 1. When coinjected with an SRE oligonucleotide, it induced c-fos expression in quiescent cells, whereas injection of SRE sequence alone failed to do so. Moreover, injection in quiescent cells of an SRE oligonucleotide together with a p-fos-lacZ construct containing the c-fos SRE as well as an in-frame insertion of FIRE resulted in a block to beta-galactosidase expression that can be relieved by coinjection of the FIRE sequence.
The c-fos proto-oncogene is rapidly and transiently induced by a variety of extracellular stimuli. We have previously shown that conditioned media from v-sis transformed NRK cells rapidly induces a DNA binding protein which binds to a conserved sequence upstream of the human c-fos gene. We now show that purified recombinant c-sis/PDGF can induce this binding activity which we have termed SIF, for sis-inducible factor. Oligonucleotides which bind to the SIF protein will confer sis/PDGF inducibility onto a truncated, unresponsive c-fos promoter. However, sequences lying between -100 and -57 of the c-fos gene are required for this induction. The sis-responsive element functions independently of a region of dyad symmetry previously identified as the serum responsive element (SRE). The time course of c-fos expression driven by the sis-responsive element is similar to that mediated by the SRE. Unlike the SRE, which can respond to signals generated by sis/PDGF, serum or phorbol esters, the SIF binding element mediates c-fos induction only in response to sis/PDGF. The SRE and SIF elements function in an additive manner to stimulate the transcription of the c-fos gene in response to sis/PDGF.
pp60c-src is phosphorylated in vivo at tyrosine 527, a residue not present in pp60v-src (its transforming homolog), and not at tyrosine 416, its site of in vitro autophosphorylation. To test the hypothesis that tyrosine phosphorylation regulates pp60c-src biological activity, we constructed and studied pp60c-src mutants in which Tyr 527 and Tyr 416 were separately or coordinately altered to phenylalanine. Tyr----Phe 527 mutation strongly activated pp60c-src transforming and kinase activities, whereas the additional introduction of a Tyr----Phe 416 mutation suppressed these activities. Tyr----Phe 416 mutation of normal pp60c-src eliminated its partial transforming activity, which suggests that transient or otherwise restricted phosphorylation of Tyr 416 is important for pp60c-src function even though stable phosphorylation is not observed in vivo.
Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.
Binding of interferons IFN-alpha and IFN-gamma to their cell surface receptors promptly induces tyrosine phosphorylation of latent cytoplasmic transcriptional activators (or Stat proteins, for signal transducers and activators of transcription). Interferon-alpha activates both Stat91 (M(r) 91,000; ref. 1) and Stat113 (M(r) 113,000; ref. 2) whereas IFN-gamma activates only Stat91 (refs 3, 4). The activated proteins then move into the nucleus and directly activate genes induced by IFN-alpha and IFN-gamma. Somatic cell genetics experiments have demonstrated a requirement for tyrosine kinase-2 (Tyk2) in the IFN-alpha response pathway and for Jak2 (ref. 6), a kinase with similar sequence, in the IFN-gamma response pathway. Here we investigate the tyrosine phosphorylation events on Stat and Jak proteins after treatment of cells with IFNs alpha and gamma and with epidermal growth factor (EGF). Stat91 is phosphorylated on Tyr701 after cells are treated with IFN-alpha and EGF, as it was after treatment with IFN-gamma (ref. 8). We find that Jak1 also becomes phosphorylated on tyrosine after cells are treated with these same three ligands, although each ligand is shown to activate at least one other different kinase. Jak1 may therefore be the enzyme that phosphorylates Tyr 701 in Stat91.