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and RIß Transcripts and Proteins in Systemic Lupus Erythematosus T Lymphocytes1
*
Section on Rheumatology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157; and
Institute of Medical Biochemistry, University of Oslo, Oslo, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and RIß, may be associated
with a diminution of RI and/or RIß protein. Sixteen SLE subjects
with a mean (±1 SD) SLE disease activity index of 12.4 ± 7.2
were studied. Controls included 16 normal subjects, six subjects with
primary Sjögren’s syndrome (SS), and three subjects with SS/SLE
overlap. RT-PCR revealed that normal, SS, SS/SLE, and SLE T cells
expressed mRNAs for all seven R and catalytic (C) subunit isoforms.
Quantification of mRNAs by competitive PCR revealed that the ratio of
RI mRNA to RIß mRNA in normal T cells was 3.4:1. In SLE T cells
there were 20 and 49% decreases in RI and RIß mRNAs (RIß;
p = 0.008), respectively, resulting in an
RI:RIß mRNA of 5.3:1. SS/SLE T cells showed a 72.5% decrease in
RIß mRNA compared with normal controls (p =
0.01). Immunoblotting of normal T cell RI and RIß proteins
revealed a ratio of RI:RIß of 3.2:1. In SLE T cells, there was a
30% decrease in RI protein (p = 0.002) and a
65% decrease in RIß protein (p < 0.001),
shifting the ratio of RI:RIß protein to 6.5:1. T cells from 25%
of SLE subjects lacked any detectable RIß protein. Analysis of
several lupus T cell lines demonstrated a persistent deficiency of both
proteins, excluding a potential effect of disease activity. In
conclusion, reduced expression of RI and RIß transcripts is
associated with a decrement in RI and RIß proteins and may
contribute to deficient type I protein kinase A isozyme activity in SLE
T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathogenic mechanism(s) leading to abnormal T cell functions in SLE remains incompletely understood. To account for these diverse T cell immune effector dysfunctions, we initially proposed that a disorder(s) primary to the T cell may exist in SLE (6, 7). We subsequently demonstrated that lupus T cells exhibit impaired cAMP-dependent, protein kinase A (PKA)-catalyzed protein phosphorylation due to a deficiency of type I PKA (PKA-I) isozyme activity (8, 9). This was the first identification of a disorder of signal transduction in human lupus T cells. To date, about 80% of unselected SLE subjects express the isozyme deficiency (10), the mean activity of which ranges from 20–30% of control values (11). Compared with healthy control T cells, PKA-I isozyme kinetics were significantly abnormal in SLE T cells, raising the possibility that a molecular disorder of the RI subunit might exist (11).
The adenylyl cyclase/cAMP/PKA pathway is a principal signal
transduction system in T cells (12). In the holoenzyme form, PKA (also
termed cAMP-dependent protein kinase) is a tetrameric serine/threonine
kinase comprising two regulatory (R) subunits and two catalytic (C)
subunits, R2C2. The PKA enzyme is comprised of
two isozymes, PKA-I and PKA-II (13, 14, 15). Cloning and sequencing of R
and C subunit genes from human tissues has led to the identification of
four R subunit isoforms, RI, RIß, RII, and RIIß (16, 17, 18, 19), and
three C subunit isoforms, C, Cß, and C
(20, 21). These isozymes
possess either a 49- to 53.5-kDa RI subunit or a 51- to
54-kDa RII subunit, respectively, and a 40-kDa C subunit (14, 15, 22).
The PKA-I isozyme can be formed by homodimerization of RI or RIß
(i.e., RI2C2 or
RIß2C2) or heterodimerization of RI and
RIß (i.e., RIRIßC2) (23). The holoenzyme can be
comprised of any C subunit isoform.
PKA is the only known intracellular receptor for cAMP in tissues (13)
and is activated by the binding of cAMP to receptor sites on the R
subunits. Receptors for cAMP on the R subunits consist of two tandem
sites, A and B, located in the carboxyl two-thirds of the molecule.
cAMP binding proceeds through the more carboxyl-terminal B site
initially, resulting in a conformational change in the R subunit that
permits subsequent binding of cAMP to the A site (24). Both isozymes
are activated by binding of cAMP to the A and B sites on R subunits,
resulting in rapid dissociation of holoenzyme as depicted by the
equation: R2C2 + 4cAMP 
R2cAMP4 + 2C (14, 15). In the T cell, ligand
binding to the TCR rapidly initiates a signal that promptly activates
the protein trysoine
kinase/polyphosphoinositide/Ca2+/protein kinase C pathway
and subsequently activates the PKA-I isozyme (25, 26). The PKA-II
isozyme is activated subsequently. Depending upon the isozyme’s
compartmentalization within the cell (27), cAMP-dependent,
PKA-catalyzed protein phosphorylation regulates diverse downstream
events, including chaperoning, cytokine production, cytotoxicity,
exocytosis, K+ channel function, and signaling pathways
(28, 29, 30, 31, 32, 33, 34, 35). By regulating these events, PKA-catalyzed protein
phosphorylation directly modulates mitosis and immune effector
functions (12).
In this analysis we tested the hypothesis that altered expression of
RI and/or RIß transcripts is associated with reduced amounts of
RI and/or RIß proteins and a consequent deficiency of PKA-I
isozyme activity in SLE T cells. Our results demonstrate that, in
contrast to both normal and primary Sjögren’s syndrome (SS)
disease controls, SLE T cells possess reduced amounts of both RI and
RIß transcripts and proteins. RIß protein is either significantly
decreased or absent in most lupus subjects. T cells from subjects with
an overlap of SS and SLE (SS/SLE) also exhibit significantly reduced
amounts of RIß transcript and protein. Thus, diminished amounts of
RI and RIß mRNA may contribute to reduced amounts of RI and
RIß proteins, resulting in a deficiency of PKA-I isozyme activity in
SLE T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixteen SLE subjects with a mean age (±1 SD) of 38.5 ± 12
yr (range, 19–66 yr) were studied. All subjects fulfilled four or more
of the criteria for the classification of SLE (36). Of these SLE
subjects, 14 were female, 13 were white and three were black, and the
mean (±1 SD) SLE disease activity index (SLEDAI) was 12.4 ± 7.2
(range, 5–32) (9, 11, 37). Sixteen healthy controls with a mean age of
37.3 ± 7.6 yr (range, 22–63 yr) were studied. Of these controls,
13 were female, and 14 were white and two were black. Six subjects with
SS with a mean age of 44.2 ± 5.0 yr (range, 28–56 yr) served as
disease controls. Of these, all subjects were white and female. Three
female subjects with an overlap between SS and SLE (SS/SLE) were
recruited for these experiments. All were female, two were Caucasian
and one was black, and their mean age was 66 ± 18.5 yr. These
subjects fulfilled criteria for both SS and SLE (36, 38). Table I
summarizes the characteristics of the SLE population,
including disease duration, SLEDAI score, and current therapy.
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10 mg/day prednisone) were entered into this protocol; these
individuals were studied 24 h after their last oral dose.
Nonsteroidal anti-inflammatory agents and hydroxychloroquine were
withheld for 72 h and 7 days, respectively, before study when
clinically feasible. Informed consent to participate in this study and to obtain peripheral venous blood by venipuncture or mononuclear leukocytes by leukopheresis was obtained from subjects and controls. The research protocols and consent forms were approved by the institutional review board of the Wake Forest University/Baptist Medical Center.
T lymphocyte isolation and phenotypic characterization
SLE and control T lymphocytes were isolated and enriched from PBMC or cells obtained by leukopheresis as previously described (11). Cytofluorographic analysis of SLE T cells revealed that enriched cells expressed a mean (±SEM) of 96 ± 1.2% CD3+.
T cell lines
PBMC from normal controls and SLE subjects were isolated. T cells were stained with propidium iodide (PI), and the proportions of cells in G0/G1, S, and G2/M phases of the cell cycle were quantified by flow cytometry. To propagate T cell lines in vitro, PBMC were cultured in RPMI 1640 and HL-1 3:1 (BioWhittaker, Walkersville, MD) supplemented with 5% FBS, 25 mM HEPES, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 IU of penicillin, and 1 µg/ml PHA. After 3 days, T lymphoblasts were passaged and cultured in the above medium further supplemented with 20 U/ml IL-2 and 40 U/ml IL-4 (Genzyme, Cambridge, MA). Concomitant stimulation of T cell lines with IL-2 and IL-4 maintained similar proportions of CD3+,CD4+ and CD3+,CD8+ subsets as those observed in freshly isolated T cells. Thereafter, T cells were passaged twice weekly. In these experiments, we did not separate CD3+ T cells into subsets. At the 10th passage, cycling T cells were harvested, and cell cycle analysis was quantified by PI. To force cells to re-enter G0/G1, T cells were transferred to RPMI 1640 supplemented with only 2% FBS, antibiotics, and L-glutamine. At 72 h, rested T cells were harvested and stained with PI, and the proportion of cells in each phase of the cell cycle was quantified. Less than 2% of control and SLE T cells underwent apoptosis during this time due to withdrawal of cytokines, as demonstrated by the absence of hypodiploid cells. Cell surface phenotyping was performed by flow cytometry.
PKA-I and PKA-II fractionation by DE52 cellulose chromatography
T cell PKA-I isozyme was fractionated by DE52-cellulose chromatography as previously described (11).
PKA assay
The PKA assay was performed as previously described (11).
Isolation of RNA and cDNA synthesis
Total cellular RNA was extracted from 10 x 106 T cells, and single-stranded cDNA (sscDNA) was synthesized from total RNA as previously detailed (25, 40). The integrity of isolated mRNA and successful cDNA synthesis were confirmed for each sample by amplifying ß-actin and RIß in the same PCR reaction, since both primer pairs have similar annealing temperatures (data not shown).
Oligonucleotide primers for PCR amplification
Oligonucleotide primers for RI, RIß, RII, RIIß, C,
Cß, and C were designed based on published sequences (16, 17, 18, 19, 20, 21)
using the Oligomer version 4.0 program and were synthesized by National
Biosciences (Plymouth, MN). The upstream and downstream primers of each
isoform are shown in Table II.
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Each reaction mixture consisted of 10% sscDNA, 25 pmol of each
primer (Table II
), 1x PCR buffer A (10 mM Tris-HCl (pH 8.3), 50 mM
KCl], 2.5 mM MgCl2, 200 µM of each dNTP, 1.25 U of Taq
polymerase (Perkin-Elmer/Cetus, Emeryville, CA), and ddH2O
in a final volume of 50 µl. The reaction mixture was subjected to 30
cycles of denaturation (94°C, 1 min), primer annealing (temperature
dependent on primer pair; cf., Table II) for 2 min, and extension for 3
min at 72°C plus 2 s added for each cycle using a DNA Thermal
Cycler (Perkin-Elmer/Cetus). Five microliters of reaction mixture was
then analyzed on a 2% agarose gel in Tris-HCl/acetate/EDTA buffer
(buffer B). One microgram of HaeIII-digested 
X174DNA
(Life Technologies, Gaithersburg, MD) were used as m.w. markers: 1353,
1078, 872, 603, 310, and 234 bp. PCR products were purified using
Wizard Plus (Promega, Madison, WI). Specific gene amplification was
confirmed by sequencing PCR products by an automated DNA sequencer (ABI
Prism 377, Applied Biosystems, Foster City, CA).
Construction of RI and RIß isoform MIMICs for competitive PCR
(C-PCR)
To quantify RI and RIß transcripts in T cells we used
C-PCR, a method that is 1,000- to 10,000-fold more sensitive than
traditional RNA blot techniques (41). Neutral DNA fragments
(BamHI/EcoRI fragment of V-erbB;
Clontech, Palo Alto, CA), 580 bp in length, were used to construct
RI and RIß isoform MIMICS. Composite primers containing RI and
RIß isoform gene-specific primer sequences in addition to 20
nucleotides that hybridize to neutral DNA fragments were designed using
the Oligomer Version 5.0 program (Table III). RI and
RIß isoform-specific MIMICs were generated by selecting a neutral DNA
fragment region containing an identical percentage of GC content as the
amplified segment of RI or RIß isoform and by using RI and
RIß isoform-specific composite and gene-specific primers in PCR
amplification. The sizes of RI and RIß MIMICs were adjusted to
353- and 535-bp lengths, respectively, by selecting appropriate
sequences along the neutral DNA fragment as the primer template to
distinguish RI and RIß isoform PCR products of 227 and 342 bp in
length, respectively. This yielded neutral DNA fragments with RI or
RIß isoform gene-specific sequences incorporated at the ends that
were used as the RI and RIß MIMICs. Molar quantities of these
MIMICs were calculated, diluted with the MIMIC dilution solution (50
µg/ml ultrapure glycogen) to a concentration of 100 attomoles/µl,
and used as stock solution for internal standards (MIMICs) for
quantification of RI and RIß isoform transcripts by C-PCR. The use
of MIMICs containing the same primer sequences as well as an identical
percentage of GC content of amplified segments of RI or RIß
isoform makes it possible to quantify transcripts of each subunit in
cDNA samples more accurately, because MIMICs and RI or RIß are
amplified with equal efficiencies. In a series of experiments, we used
the following concentrations of RI and RIß isoform MIMICs, as
shown in Table IV, as internal standards along with equal quantities of
cDNA samples, because the RI and RIß transcripts were within this
range.
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and RIß transcripts in SLE and control
subjects
Twofold dilutions of MIMICS were spiked into PCR reaction tubes
containing equal amounts of cDNA samples (10% of sscDNA synthesized
from 1 µg of total cellular RNA) from control or SLE T cells and
amplified as described above. Amplified RI or RIß isoform and
their respective MIMICs were distinguished on ethidium bromide-stained
agarose gels by discrete base pair lengths of MIMIC, RI, and RIß
isoform-specific PCR products. Polaroid (Cambridge, MA) photographs of
gels were obtained with a UV transilluminator. The amounts of RI and
RIß isoform transcripts were estimated in SLE and control samples by
comparison with different concentrations of known standard (MIMIC) and
identifying which concentration of standard matched with the gene
product. Transcripts were expressed as attomoles per microgram of total
RNA (26).
Immunoblotting
Whole T cell lysate lacking nuclei was prepared as previously described (26). In summary, T cells were lysed in 300 µl of buffer C (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin) by sonication. Samples were centrifuged at 10,000 rpm at 4°C for 5 min in a microcentrifuge to eliminate nuclei and debris, supernatants were separated, and the protein content was quantified (42). One hundred and fifty micrograms of total protein from each sample was separated by 10% one-dimensional SDS-PAGE, and the proteins were then transferred to Immobilon-P (Millipore, Bedford, MA). Membrane was blocked with Blotto for enhanced chemiluminescence (ECL)-based detection for 1 h. The membranes were immunoblotted with a 1/1000 dilution of anti-RI mAb (Transduction Laboratories, Lexington, KY) or a 1/25 dilution of polyclonal rabbit anti-human RIß (23), washed with buffer D (100 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 0.1% Tween-20), and probed with a 1/4000 dilution of horseradish peroxidase-labeled sheep anti-mouse or a 1/4500 dilution of peroxidase-conjugated anti-rabbit IgG diluted in Blotto. After washing four times with buffer D, the blots were developed by ECL reagent. Primary and secondary Abs were then extracted from the membrane using buffer E (62.5 mM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS) and were reprobed with a 1/100 dilution of polyclonal rabbit anti-human actin, a 1/4000 dilution of horseradish peroxidase-labeled sheep anti-mouse IgG, and ECL. Quantification of protein was performed by laser densitometry and was expressed as arbitrary units (AU).
Quantification of RI and RIß proteins in total T cell protein was
derived from reference standard curves. To generate standard curves,
increasing concentrations of T cell protein ranging from 25–200 µg
were separated by one-dimensional SDS-PAGE, transferred to Immobilon-P,
and immunoblotted with anti-RI mAb. Immunoblots were developed with
ECL for 1 min and scanned by laser densitometry, and linear curves were
plotted. Because T cell protein from a normal subject was run with each
SLE sample, the use of a reference standard curve permitted
normalization of samples and eliminated any potential interexperiment
variability that might occur with the ECL reagent. Quantification of
RI and RIß proteins was expressed as AU.
Statistical analysis
Statistical significance (p 0.05) was
calculated by Student’s t test, Mann-Whitney U
rank-sum test, and/or ANOVA (SigmaStat, Jandel Scientific, Corte
Madera, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total RNA was isolated from the T cells of 16 SLE subjects, three
SS/SLE subjects, 16 healthy controls, and six SS disease controls,
respectively. The integrity of mRNAs was verified for each sample by
monitoring ß-actin and RIß; no degradation was detected. Using
RT-PCR, we amplified cDNAs for the PKA RI, RIß, RII, RIIß,
C, Cß, and C isoforms from each donor using the primer pairs
shown in Table II. T cells from SLE subjects as well as healthy and
disease controls expressed transcripts for each of the seven R and C
subunit isoforms (Fig. 1, SLE and healthy controls only).
The electrophoretic mobility of each isoform PCR product was consistent
with its expected base pair length (Table II). Controls in which PCR
was performed in the absence of either RT or template showed
no R or C isoform transcripts, excluding DNA contamination of RNA.
Direct sequencing of the PCR products by an automated DNA
sequencer revealed identity with the expected human PKA cDNA sequences,
as determined from the GenBank-EMBL DNA database (data not shown).
These results demonstrate that T cells from both control groups as well
as lupus subjects constitutively express transcripts of all known PKA R
and C isoforms.
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and RIß transcripts in SLE and control
T cells
We used C-PCR to quantify RI isoform transcripts. The mean
concentration (±SEM) of RI transcripts in normal T cells was
0.76 ± 0.12 attomoles/µg total RNA (median, 0.73; 25–75th
percentile, 0.49–0.98 attomoles/µg RNA). The mean concentration of
RI transcript in SS T cells was 0.98 ± 0.002 attomoles/µg
total RNA (median, 0.98; 25–75th percentile, 0.82–1.1). By contrast,
the mean content of RI transcripts in SLE T cells was 0.61 ±
0.09 attomoles/µg RNA (median, 0.49; 25–75th percentile, 0.36–0.98
attomoles/µg RNA). Although SLE T cells had a 20% reduction in the
mean content of RI transcripts, this was not significantly different
from control levels (p = 0.36; Fig. 2 and Table V).
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). By contrast, the mean
concentration of RIß isoform mRNAs in SLE T cells was 0.11 ±
0.02 attomoles/µg RNA (median, 0.09; 25–75th percentile, 0.06–0.12
attomoles/µg RNA). Compared with normal controls, this 49% reduction
in the content of RIß isoform transcripts in SLE T cells was
statistically significant (p = 0.008; Fig. 2
and Table IV). These data establish that the amount of RIß transcript
is significantly reduced in SLE T cells.
Based upon these data, we estimated that the ratio of RI:RIß mRNA
is 3.4:1 in normal T cells. In SS T cells, the greater amount of RI
mRNA results in an increased ratio of RI:RIß mRNA of 4.4:1.
Because of the altered content of both RI and RIß transcripts in
SLE T cells, the ratio of RI:RIß mRNA is increased to 5.3:1 (Table V).
An overlap of SS and SLE occurs in a proportion of affected subjects.
To determine whether individuals with an SS/SLE overlap exhibit altered
amounts of RI and/or RIß transcripts, we studied T cells from
three such persons. The mean RI mRNA content was 1.06 ± 0.4
attomoles/µg RNA, a value that is not significantly increased over
that observed in normal and SS controls or SLE subjects. The RIß mRNA
content was 0.06 ± 0.008 attomoles/µg RNA, a value that is
statistically reduced compared with that in normal controls
(p = 0.01), but is not significantly different
from that observed in SLE (Table IV). Because of the increased content
of RI and markedly reduced amount of RIß transcripts, the ratio of
RI:RIß mRNA is a striking 17.3:1. However, like SLE T cells,
SS/SLE overlap T cells exhibit significantly reduced amounts of RIß
transcript compared with normal controls.
T cell RI and RIß protein content in SLE and controls
Altered transcription of both RI isoforms in lupus T cells
prompted us to quantify the contents of RI and RIß isoform
proteins in T cell lysates. Whole T cell lysates were separated by
one-dimensional SDS/PAGE, electroblotted onto membrane, and
immunoblotted with anti-RI mAb, and the protein was quantified by
laser densitometry. This mAb recognizes a common epitope of the
carboxyl terminus of both RI isoforms. Fig. 3A shows a representative immunoblot of T cell
lysates from six healthy controls. Based on 16 samples, the mean
(±SEM) concentration of RI protein was 11.35 ± 0.25 AU
(median, 11.23; 25–75th percentile, 10.99–12.03). The mean
concentration of RIß protein was 3.59 ± 0.29 AU (median, 3.35;
25–75th percentile, 3.16–3.67). The ratio of RI:RIß protein in
normal T cells is 3.2:1, a value similar to the ratio of RI:RIß
mRNA (Table V). This result suggests that there is approximately a 1:1
relationship between the ratio of RI and RIß isoform transcripts
and proteins.
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B is a representative immunoblot of T cell lysates
from three SS controls and three SS/SLE overlap subjects. The mean
concentration of RI protein in six SS T cells was 12.77 ± 0.57
AU (median, 12.97; 25–75th percentile, 12.01–13.48). The mean
concentration of RIß protein in six SS T cells was 2.99 ± 0.71
AU (median, 2.34; 25–75th percentile, 2.26–3.89). Thus, the ratio of
RI:RIß protein in SS controls was 4.3:1, similar to the ratio of
RI:RIß mRNA (Table V). This result suggests that there is
approximately a 1:1 relationship between the ratio of RI
and RIß isoform transcripts and proteins in SS control T cells.
In contrast to controls, SLE T cells had reduced amounts of both RI
and RIß proteins. Of 16 specimens, 14 had reduced amounts of RI
isoform, and 9 had diminished content of RIß isoform. No RIß
isoform protein was detected in four of 16 specimens (25%). Fig. 3C shows the immunoblots of T cells from six representative
SLE subjects. Of these, RI protein was reduced in four subjects and
was physiologic in two subjects. RIß protein was undetectable in two
subjects, was reduced in three subjects, and was physiologic in one
subject. In 16 SLE T cell samples, the mean concentration of RI
protein was 8.06 ± 0.6 AU (median, 8.14; 25–75th
percentile, 7.25–9.36), and the mean content of RIß was
1.25 ± 0.34 AU (median, 1.1; 25–75th percentile, 0.08–2.38;
Table IV). Compared with normal controls, there was a 30% decrease in
RI protein (p = 0.002) and a 65% decrease
in RIß protein (p < 0.001), shifting the
ratio of RI:RIß protein to 6.5:1 (Table V). This ratio was
increased over the ratio of RI:RIß mRNA in SLE T cells, raising
the possibility that post-translational degradation of RIß isoforms
may also be occurring. Moreover, compared with SS controls, the amounts
of RI and RIß isoform proteins in SLE T cells were also
significantly reduced (RI, p < 0.002; RIß,
p = 0.034; Table IV). These findings suggest that SLE T
cells possess significantly reduced amounts of both RI isoforms, but
that the amount of RIß protein is proportionally more diminished.
The reduction of both RI and RIß isoform proteins in the T cells
of this cohort of SLE subjects differs from our previous
findings. We previously reported that the amount of RI
protein in SLE T cells was comparable to that of normal T cells (11).
This apparent discrepancy derives from our use in those experiments of
an anti-RI isoform Ab to immunoblot only 20 µg of T cell
lysate protein/lane (11). Using an anti-RI mAb directed against
both RI isoforms, we subsequently established that it is necessary to
use at least 150 µg of protein/lane to identify both isoforms
consistently. The use of low amounts of protein did not provide
sufficient sensitivity to discriminate modest alterations in RI
isoform protein content. Thus, our recognition herein that both RI
and RIß isoform proteins are reduced in SLE T cells supersedes our
previous report.
To determine whether the markedly altered ratio of RI:RIß
transcripts in SS/SLE overlap T cells was associated with aberrant
protein expression, we quantified RI and RIß protein contents
(Fig. 3B). The mean content of RI isoform in SS/SLE
overlap T cells was 10.72 ± 0.59 AU (median, 10.73; 25–75th
percentile, 9.96–11.49), and the mean RIß concentration was 0.9
± 0.09 AU (median, 0.98; 25–75th percentile, 0.79–1.002). Thus, the
ratio of RI:RIß in the SS/SLE overlap T cells was 11.9:1, which
contrasts sharply with that observed in normal or SS control T cells
and largely reflects the markedly reduced RIß isoform content.
Although there are no significant differences between the amount of
RI protein in normal or SS control T cells and SS/SLE T cells, there
is a significantly reduced amount of RIß protein in SS/SLE T cells
compared with either normal T cells (p = 0.024)
or SS T cells (p = 0.043; Fig. 3B
and Table V). As anticipated, SLE T cells have a significantly lower
amount of RI protein than SS/SLE T cells (p
= 0.05;Table IV), although the amounts of RIß isoform protein in SLE
and SS/SLE T cells are not significantly different. Thus, when the
amounts of the RI proteins in SS/SLE and SLE T cells are compared, it
would appear that both SLE and SS/SLE T cells manifest only a
significant deficiency of RIß isoform protein.
Analysis of SLE T cell lines
T cell lines were established in four SLE subjects whose cells did
not express the RIß isoform. Four T cell lines derived from healthy
controls were initiated concomitantly. Before stimulation, 99% of
control and patient T cells were in G0/G1 of
the cell cycle, respectively. After 10 passages, cycling T cells were
harvested, the proportion of cells in each phase of the cell cycle was
quantified, and RI immunoblots were performed on T cell lysates. The T
cell lines were then cultured in RPMI 1640 medium containing 2% FBS in
the absence of PHA and cytokines to induce cells to revert to
G0/G1. After 72 h, the proportion of
rested, viable cells in each phase of the cell cycle was quantified,
and RI immunoblots were performed on T cell lysates. Fig. 4
shows the results of a representative experiment. Freshly isolated
normal control T cells possessed both RI and RIß isoforms, whereas
SLE cells exhibited only the RI isoform. After 10 passages, neither
control nor patient T cells expressed the RIß isoform in cycling
cells. The absence of RIß may be the result of preferential, rapid
degradation by proteolysis during rapid cell proliferation due to
persistent enzyme activation and utilization (data not shown). At this
time, 29 and 34% of control and lupus T cells, respectively, were in S
phase of the cell cycle. After resting cells for 72 h, the
proportions of control and SLE T cells in S phase had dropped to
<10%, and the RIß protein was re-expressed in control, but not SLE,
T cells. These results suggest that although PKA-I is activated (25),
and RIß may be rapidly degraded during T cell activation and
proliferation, RIß protein reaccumulates when control cells return to
G0/G1. By contrast, the RIß protein failed to
be re-expressed in viable lupus T cells. Failure to re-express RIß
was not due to apoptosis, because 
98% of cells were viable, and only
2% were hypodiploid by PI staining. Because lupus T cell lines that
had returned to G0/G1 faithfully recapitulated
the absence of the RIß protein in freshly isolated cells, failure to
express the protein is unlikely to be secondary to any impact of
clinical disease activity on T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and RIß
transcripts are reduced compared with those in control T cells. Indeed,
the amount of the RIß transcript is significantly diminished. Lastly,
the amounts of both RI and RIß proteins in SLE T cells are
significantly decreased compared with those in control T cells. Taken
together, these data support the hypothesis that altered expression of
RI and RIß transcripts is associated with reduced amounts of RI
and RIß proteins. This reduction of both RI and RIß proteins may
contribute to the observed deficiency of PKA-I isozyme activity in SLE
T cells.
All mammalian cells, including human T lymphocytes, express both the
PKA-I and PKA-II isozymes (22, 43, 44). It is currently held that
RI, RII, and C isoforms are expressed ubiquitously in tissues,
whereas RIß, RIIß, Cß, and C isoforms appear to be restricted
to brain, ovary, and/or testis (16, 17, 21, 45, 46, 47, 48). In the human T
cell, only transcripts for the RI, RII, C, and Cß subunits
have been previously identified by Northern blotting (34, 44). However,
using RT-PCR, a sensitive and specific method to detect gene
transcripts (49), we have identified constitutively expressed mRNAs for
all seven known RI, RII, and C isoforms of PKA in normal, SS, SLE, and
SS/SLE T cells. During the performance of RT-PCR, the use of controls
deleting either reverse transcriptase or template excluded potential
contamination of RNA by DNA and/or other cDNAs, thereby verifying the
presence of the transcripts in T cells. Thus, this observation verifies
our recent identification of all the R and C transcripts in a human
CD4+ leukemic T cell line (35). However, this is the first
demonstration that normal human T cells also express all R and C
subunit isoform transcripts. This observation suggests that the ß
isoforms of the R and C subunits and the isoform of the C subunit
are not restricted to the nervous system, ovary, or testis. Moreover,
the identification of constitutively expressed R and C isoform
transcripts in SLE T cells demonstrates that these genes are being
transcribed.
We have previously demonstrated that T cells from subjects with SLE
exhibit a disorder of signal transduction involving the adenylyl
cyclase/cAMP/PKA pathway (6). This signaling disorder is characterized
by impaired cAMP-dependent, PKA-catalyzed protein phosphorylation due
to a deficiency of PKA-I phosphotransferase activity (8, 9). This was
the first demonstration of an abnormal signaling pathway in SLE T
cells. We now estimate that T cells from about 80% of SLE subjects may
harbor the PKA-I isozyme deficiency (10). Analysis of isozyme kinetics
in SLE T cells with deficient PKA-I activity demonstrated a 2.2-fold
increase in the Michaelis-Menten constant (Km),
a 2.5-fold increase in the apparent cAMP half-maximal activation
(Ka) of holoenzyme, a 3.8-fold decrease in the
maximal velocity (Vmax), and a 2-fold reduction
in the maximal binding of cAMP (Bmax) to the RI
subunit compared with control T cells (11). These abnormal isozyme
kinetics raised the possibility that a structural defect in a gene(s)
encoding RI and/or RIß isoform proteins may exist (50).
To quantify the amounts of RI and RIß transcripts in control and
SLE T cells, we used C-PCR. When compared with normal control T cells,
there were mean 20 and 49% reductions in the amounts of RI and
RIß mRNAs in SLE T cells, respectively. In contrast, T cells from SS
disease controls expressed higher amounts of RI transcript than
normal T cells. Although the reduced content of RI transcripts in
SLE T cells did not differ statistically from that of normal control T
cells, it may be functionally relevant and contribute to diminished
RI protein generation. By contrast, SLE T cells possessed
significantly less RIß mRNA. This decrement in both transcripts led
to an upward shift of the ratio of RI:RIß transcripts in SLE T
cells to 5.3:1 from 3.4:1 in normal T cells. Interestingly, T cells
from subjects with a SS/SLE overlap disorder exhibited a chimeric
pattern: high RI mRNA (observed in SS) and low RIß mRNA content
(observed in SLE), resulting in an exceptionally elevated RI:RIß
ratio of 17.3:1. Thus, T cells from overlap subjects also express the
markedly reduced RIß mRNA content observed in SLE. Considering the
magnitude of the decline in RIß mRNA in both SLE and SS/SLE T cells,
we propose that transcriptional dysregulation of the RIß gene may
exist. Because aberrant transcription of this isoform gene could
contribute to the observed deficiency of both RI and RIß isoform
proteins and, therefore, to the deficiency of PKA-I isozyme activity,
identifying the nature of the putative transcriptional dysfunction will
be crucial.
Considering the complexities of RI transcriptional regulation, we
propose four potential mechanisms that could, singly or combined,
account for the observed reductions in RI and RIß transcripts.
First, because T cells from all lupus subjects express transcripts for
both RI isoforms by RT-PCR, it is conceivable that there may be
impaired RI transcription initiation. An RI genomic mutation(s) could
contribute to such a disorder (50, 51). Second, altered RI isoform
transcriptional rates and/or transcript stabilities could contribute to
the observed reduction of RI transcripts. Third, impaired
phosphorylation of a transcription factor(s) may affect the capacity of
the underphosphorylated factor(s) to bind to its target DNA motif,
thereby hindering transcription initiation. We have previously
postulated that a principal complication of a PKA isozyme deficiency
could be impaired phosphorylation of either downstream protein kinase
intermediates required to phosphorylate trans-activating
factors or, perhaps, direct underphosphorylation of such
trans-activation factors (52). In either case,
underphosphorylation may prevent the necessary conformational changes
for optimal DNA binding, leading to an inefficient protein/DNA
interaction (52). Finally, a mutation(s) within a DNA recognition
sequence(s) of a transcription factor(s) could also impede optimal DNA
binding and, therefore, result in defective transcription initiation.
These putative mechanisms are currently being investigated in SLE T
cells.
We observed an approximately 1:1 relationship between the ratios of RI
isoform transcripts and proteins in both normal and SS T cells. This
finding suggests that there is a direct relationship between
transcription and translation of RI and RIß in normal and SS
control T cells. By contrast, SLE T cells possessed a mean 30% less
RI protein and 65% less RIß protein, shifting the ratio of
RI:RIß protein to 6.5:1. Interestingly, in contrast to normal and
disease controls, RIß protein was undetectable in four of 16 lupus T
cell lysates. Such a finding is unlikely to be accounted for on the
basis of either clinical disease activity or technical errors for three
reasons. First, we have recently completed an analysis of PKA-I
activities in 35 SLE subjects that revealed no apparent relationship
between low PKA-I activities and clinical disease activity. This
apparent lack of relationship between clinical disease activity and
deficient PKA-I activity was verified in an analysis of 15 SLE subjects
over a 4-yr period (10). Second, repetitive analyses of freshly
obtained T cells from these subjects over a period of 12 mo revealed
persistently undetectable RIß protein. Finally, analysis of SLE T
cell lines propagated 10 passages in vitro and subsequently returned to
the G0/G1 phase of the cell cycle revealed
persistently undetectable RIß protein in viable T cells, excluding a
potential effect of disease activity. This contrasts with control T
cell lines in which the RIß subunit was re-expressed upon reverting
to G0/G1 from S phase. Thus, T cells from a
majority of SLE subjects exhibit disordered regulation of PKA RI
proteins. Whether this is a heritable trait is currently being studied.
Long-standing evidence in multiple human cell types suggests that the
RI2C2 and RIß2C2
isozymes regulate cell growth (53). In human T cells, the PKA-I isozyme
appears to be associated with the plasma membrane fraction (22). During
the early events of TCR-dependent cellular activation, the PKA-I
isozyme is rapidly activated and phosphorylates multiple plasma
membrane-associated proteins (25, 26), including two recently
identified substrates, heat shock protein 60 and histone 2B (35).
Although RI and RIß proteins are 80–90% identical at
the amino acid level, the RIß2C2 holoenzyzme
is half-maximally activated at a 2.1-fold lower concentration of cAMP
compared with the RI2C2 holoenzyme (54, 55),
suggesting that RIß2C2 may be activated
before RI2C2. Moreover,
RIß2C2 may phosphorylate substrates distinct
from those of RI2C2 during various cellular
functions. Therefore, the reduction or absence of
RIß2C2 homodimers or RIßC2
heterodimers might be expected to significantly impair the functions of
those proteins dependent upon phosphorylation by
RIß2C2 for activation/inactivation of their
cellular functions. Furthermore, the absence of RIß protein may
contribute to the observed differences in Ka between SLE
and controls (11). Our data support the idea that reduced RI and
RIß proteins contribute to deficient PKA-I isozyme activity in SLE T
cells. The potential impact of this deficiency on T cell metabolism
and, ultimately, on various immune effector functions, such as
cytotoxicity/suppression and helper activity, in lupus pathogenesis
will be important to establish.
Although altered transcription leading to deficient synthesis of the RI
isoform proteins appears to be operative, our data raise the
possibility that post-translational mechanisms could also contribute to
deficient RI and RIß protein content in SLE T cells. First,
kinetic analyses of PKA-I activity in SLE T cells revealed significant
variances of the Vmax,
Ka(cAMP), and Bmax(cAMP)
compared with control PKA-I kinetics, suggesting the existence of a
structural abnormality(s) of a RI and/or RIß protein(s) (11).
Second, in these experiments we observed that T cells from all SLE
subjects expressed RI and RIß transcripts by PCR, but four had no
detectable RIß protein, and nine had reduced amounts of the isoform
by immunoblotting. These variances created markedly altered ratios of
RI:RIß proteins compared with control T cells, sometimes, as with
the SS/SLE overlap T cells, far greater than anticipated. Third, if
structurally abnormal RI proteins are being produced, they may undergo
proteolysis. Augmented degradation of RI and RIß proteins
resulting from enhanced proteolysis could contribute to the skewed
balance of these isoforms. PKA R subunits contain two N-terminal
sequences that can be cleaved by proteases (56). One 30-aa region is
rich in proline, glutamic acid, serine, and threonine (PEST) residues.
The PEST domain is a signature of proteins that undergo rapid
proteolysis (57). A second domain, termed the hinge region, possesses
dibasic amino acids, is especially susceptible to proteolysis in vitro,
and is immediately adjacent to the PEST domain. This region may be a
substrate for trypsin-like serine proteases and calpains (58). More
recently, PKA R subunits from both Aplysia californica and
mammals were found to be degraded by an ATP-ubiquitin-ligase-dependent
process through the 26S proteasome (59, 60). Because the roles of
proteases and the ubiquitin-proteasome pathway in the turnover of RI
isoforms have not yet been explored in human T cells, it might be
speculated that enhanced degradation of RI isoforms in SLE T cells
could account in part for the depletion of these proteins and
contribute to deficient PKA-I activity.
In summary, cAMP-dependent, PKA-catalyzed protein phosphorylation is
markedly impaired in SLE T lymphocytes due to deficient PKA-I isozyme
activity (8, 9). To our knowledge, this is the first identification of
reduced amounts of RI and RIß transcripts in a human disease.
Altered transcription of both RI isoform mRNAs, particularly the RIß
transcript, may contribute to the markedly reduced amounts of both
RI and RIß proteins and result in diminished formation of the type
I tetrameric holoenzyme, R2C2. This PKA-I
isozyme deficiency may contribute to a disorder of signal transduction
and, therefore, to a primary T cell disorder in SLE.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dama Laxminarayana, Section on Rheumatology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1058. E-mail address:
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; PKA, protein kinase A; PKA-I/II, type I/II isozyme of PKA; RI/ß, /ß isoform of regulatory subunit of PKA-I; R subunit, regulatory subunit; C subunit, catalytic subunit; RI/ß2C2, homodimer of RI or RIß isoform with C subunit; RI2C2/RIß2C2, heterodimer of RI and RIß isoforms with C subunit; SS, Sjögren’s syndrome; SS/SLE, SS/SLE overlap; SLEDAI, SLE disease activity index; PI, propidium iodide; C-PCR, competitive PCR; ECL, enhanced chemiluminescence; AU, arbitrary units; Ka(cAMP), half-maximal activation of purified holoenzyme by cAMP; Vmax, maximal velocity; Bmax(cAMP), maximal binding of cAMP; PEST, proline, glutamic acid, serine, and threonine.
Received for publication November 9, 1998. Accepted for publication February 16, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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of cAMP-dependent protein kinase encodes an alternate amino-terminal region. FEBS Lett. 246:57.[Medline]
. Nucleic Acids Res. 16:8189.) from human testis-representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase. Mol. Endocrinol. 4:465.-RIß heterodimeric complexes. J. Biol. Chem. 268:21276. subunit in T-cell receptor-triggered T-lymphocytes effector functions. J. Biol. Chem. 267:25256. of cyclic AMP-dependent protein kinase inhibits TCR-triggered secretion of IL-2 by T helper hybridoma cells. J. Immunol. 158:171.[Abstract]
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