(Received for publication, September 23, 1994; and in revised form, November 2, 1994)
From the
Proteolytic processing of select constituents of the nuclear
factor B (NF-
B)/inhibitor
B
(I
B) transcription
factor system plays an important role in regulating the biological
responses of monocytes to pro-inflammatory mediators. Nuclear
translocation of NF-
B is preceded by the proteolytic degradation
of I
B
, an ankyrin motif-rich inhibitor that traps NF-
B
in the cytoplasm. In addition, formation of cytoplasmic
NF-
B/I
B
complexes in quiescent cells requires
constitutive proteolytic processing of p105, another ankyrin motif-rich
inhibitory protein from which the p50 subunit of NF-
B is
generated. We have demonstrated that, following stimulation of human
monocytic cells with lipopolysaccharide or tumor necrosis factor-
,
this critical p105 processing event is up-regulated in concert with the
inactivation of I
B
. Moreover, the degradative loss of both
p105 and I
B
is prevented in cells depleted of intracellular
ATP. In activated monocytes, however, I
B
degradation occurs
more rapidly than p105 processing to p50. Together these findings
provide direct biochemical evidence that p105 and I
B
are
differentially sensitive targets for inducible proteolysis via
ATP-dependent degradative pathways.
The NF-B(
)/Rel family of transcription factors
participates in the induced expression of a diverse set of cellular and
viral genes that are activated in response to immune and inflammatory
signals. This set includes cellular genes that encode cytokines, cell
adhesion molecules, and procoagulant factors. In addition, NF-
B is
involved in the initiation of transcription from viral promoters that
control the expression of human immunodeficiency virus 1 and
cytomegalovirus (see (1) for review). Typically, NF-
B
resides in the cytoplasm as a ternary complex composed of two DNA
binding subunits, termed RelA (p65) and NF-
B1 (p50), bound to an
ankyrin motif-rich inhibitor called I
B
(see (2) for
review). This labile inhibitory protein is rapidly degraded in response
to multiple cellular activation signals, thereby permitting nuclear
expression of the functional NF-
B p50/RelA complex (see (3) for review).
An unusual feature of the NF-B1 gene
encoding p50 is its capacity to specify a larger precursor protein,
termed p105(4, 5, 6, 7) . This
precursor subunit has several key properties in common with
I
B
. (i) p105 resides exclusively in the cytosolic compartment
independent of the status of cellular
activation(8, 9) ; (ii) p105 contains an ankyrin
motif-rich carboxyl-terminal domain that inhibits the nuclear
expression and DNA binding activity of its amino-terminal
half(8, 9, 10, 11, 12) ;
this I
B
-related domain is constitutively degraded in order to
generate the p50 DNA binding subunit of NF-
B(13) ; (iii)
p105, like I
B
, physically sequesters RelA in the cytoplasmic
compartment (14) ; and (iv) the inducible genes encoding p105
and I
B
both contain functional NF-
B binding
sites(15, 16, 17, 18) . These
findings point to a dynamic relationship between p105/p50,
I
B
, and RelA at the protein and DNA levels that define at
least two distinct autoregulatory mechanisms for NF-
B-directed
transcription(1, 3) .
Despite these structural and
functional similarities of p105 and IB
, the proteolytic
mechanisms that control their inhibitory activities remain ill defined.
Here we demonstrate that treatment of human monocytic cells with either
bacterial lipopolysaccharide (LPS) or tumor necrosis factor-
(TNF)
stimulates p105 processing to p50 via an ATP-dependent pathway.
Moreover, agonist-induced degradation of the structurally related
I
B
protein, which occurs with more rapid kinetics relative to
p105 processing, is also regulated by an ATP-dependent process. These
findings suggest that proteolytic processing of p105 and I
B
in monocytic cells may be differentially induced during the course of
an inflammatory reaction.
Figure 1:
Effect of LPS activation on p105 and
nuclear NF-B expression in human monocytic cells. THP-1 cells were
treated with CHX (10 µg/ml), LPS (10 µg/ml), or a combination
of CHX and LPS at these concentrations for the times indicated. In CHX
+ LPS treatment, CHX was added 30 min prior to LPS. Cytosolic
extracts were subjected to immunoblot analyses using p105-specific
(amino acids 947-969) antipeptide antibody. Arrows indicate the positions of the major forms of p105-specific
polypeptides. The minor immunoreactive species migrating below the
major p105-specific polypeptides is also eliminated in blocking studies
with p105-specific (amino acids 947-969) peptides. Nuclear
extracts from the same cells were assayed for NF-
B DNA binding
activity by gel shift analyses. A composite of the resultant
B-specific nucleoprotein complexes are
shown.
Figure 2:
Pulse-chase analysis of p105 in
unstimulated and LPS- or TNF-stimulated THP-1 cells. a, a
representative pulse-chase experiment performed as described under
``Materials and Methods.'' C-Labeled molecular
weight standards (Amersham Corp.) are indicated in kilodaltons (kDa). b, quantitative phosphorimager analysis of pulse-chase data
from five independent experiments. Each point represents the mean
(± S.E.) of radiolabeled p105 relative to that detected in
pulse-labeled cells (arbitrarily set as 100%) at time =
0.
Figure 3:
Pulse-chase analysis of p50, p105, and
IB
. a, THP-1 cells were pulse-labeled (lanes 1 and 5) with
[
S]methionine/[
S]cysteine
and chased either in the absence (lanes 2-4) or presence (lanes 6-8) of LPS (10 µg/ml) for the indicated
times. Whole cell extracts were immunoprecipitated with
p105/p50-specific (amino acids 1-21) antipeptide antibody,
fractionated on SDS-polyacrylamide gels, and visualized by
fluorography.
C-Labeled molecular weight standards are
indicated in kilodaltons (kDa). p105, p50, and I
B
proteins
are indicated by arrows. b, quantitative
phosphorimager analysis of pulse-chase data from three independent
experiments. Each point represents the mean (± S.E.) of each
radiolabeled protein relative to that detected for that protein in
pulse-labeled cells (arbitrarily set as 0% for p50 and as 100% for p105
and I
B
) at time = 0.
Figure 4:
Effect of intracellular ATP depletion on
p105 proteolysis and nuclear NF-B expression in LPS-stimulated
monocytes. THP-1 cells were depleted of intracellular ATP for 2 h (see
``Materials and Methods'') and then cultured either in the
presence (lower panels) or absence (upper panels) of
LPS (10 µg/ml). As a control, cells replete with ATP were
stimulated with LPS (10 µg/ml; middle panels). Cytosolic
extracts were subjected to immunoblot analyses using p105-specific
antipeptide antibody. Nuclear extracts from the same cells were assayed
for NF-
B DNA binding activity by gel shift analyses. A composite
of the resultant
B-specific nucleoprotein complexes is
shown.
Figure 5:
IB
proteolysis in stimulated and
ATP-depleted monocytes. a and b, immunoblot analyses
using I
B
-specific (amino acids 289-317) antipeptide
antibody were performed on cytosolic extracts from THP-1 cells treated
with cycloheximide (10 µg/ml), lipopolysaccharide (10 µg/ml),
tumor necrosis factor-
(100 units/ml), or the indicated
combinations of these reagents. CHX was added 30 min prior to the
addition of agonists. c and d, immunoblot analyses
using I
B
-specific antipeptide antibody were performed on
cytosolic extracts from THP-1 cells that were depleted of intracellular
ATP and cultured either in the absence (-ATP; upper
panels) or presence (lower panels) of either LPS (10
µg/ml) or TNF (100 units/ml) for the indicated times. In control
experiments, cells replete with ATP were stimulated with either LPS (10
µg/ml) or TNF (100 units/ml) (middle
panels).
These studies with human monocytic cells demonstrate that
proteolytic processing of p105 is stimulated significantly in response
to the pro-inflammatory agents LPS and TNF. As such, inducible
proteolysis regulates not only the level of IB
in activated
monocytes(19, 23, 24) , but also the levels
of p105 and p50. These findings are in accord with some (25, 26) but not all (27) reports regarding
the regulation of p105 in other experimental systems. The rate of p105
processing may therefore be influenced in an agonist- and/or cell
type-dependent manner. We have also shown that the in vivo processing of p105 in human monocytic cells is ATP-dependent, thus
extending the in vitro findings of Fan and Maniatis (13) . In this regard, evidence is emerging that p105 and
I
B
are both phosphorylated during cellular
activation(19, 26, 28) . However, the
relationship between phosphorylation and degradation remains unknown.
Notwithstanding this uncertainty, the critical requirement for ATP in
p105 processing documented in our studies is fully consistent with
recent findings that the 26S proteasome specifically mediates this p105
processing event(29) .
Our results provide further evidence
that the NF-B transcription factor system can be regulated at
multiple levels through the induced proteolysis of I
B inhibitory
subunits and NF-
B precursor proteins. The different rates observed
for induced I
B
and p105 proteolysis in these studies imply a
bimodal function for proteolysis, namely to permit nuclear
translocation of NF-
B and to supply a second wave of p50 subunits.
This second wave could include p50 homodimers, which have been reported
to repress gene transcription in a LPS-tolerant monocytic cell
line(30) . In addition, the dilatory generation of p50 in
association with RelA could provide a mechanism to replenish the
cytoplasmic pool of preformed NF-
B that is released from
I
B
and rapidly mobilized to the nucleus.