| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
, and I
B Kinases 1 and 2 Are Downstream Effectors of CD44 During the Activation of NF-B by Hyaluronic Acid Fragments in T-24 Carcinoma Cells1
Department of Biochemistry and Biotechnology Institute, Trinity College, Dublin, Ireland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B. HA fragments activated
NF-B in the cell lines T-24, HeLa, MCF7, and J774. Further studies
in T-24 cells demonstrated that HA fragments also induced IB
phosphorylation and degradation, B-linked reporter gene expression,
and ICAM-1 promoter activity in an NF-B-dependent manner. The effect
of HA was size dependent as neither disaccharide nor native HA were
active. CD44, the principal cellular receptor for HA, was critical for
the response because the anti-CD44 Ab IM7.8.1 blocked the effect on
NF-B. HA fragments activated the IB kinase complex, and the
effect on a B-linked reporter gene was blocked in T-24 cells
expressing dominant negative IB kinases 1 or 2. Activation of
protein kinase C (PKC) was required because calphostin C inhibited
NF-B activation and IB phosphorylation. In particular, PKC
was required because transfection of cells with dominant negative
PKC blocked the effect of HA fragments on B-linked gene
expression and HA fragments increased PKC activity. Furthermore,
damnacanthal and manumycin A, two mechanistically distinct inhibitors
of Ras, blocked NF-B activation. Transfection of T-24 cells with
dominant negative Ras (RasN17) blocked HA fragment-induced B-linked
reporter gene expression, and HA fragments activated Ras activity
within 5 min. Taken together, these studies establish a novel signal
transduction cascade eminating from CD44 to Ras, PKC, and IB
kinase 1 and 2. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and insulin-like growth
factor in murine bone marrow-derived macrophages (8). HA
fragments have also been shown to elicit the expression of a
number of proinflammatory chemokines (9, 10, 11), inducible NO
synthase (9, 12), and macrophage metalloelastase
(13) through a mechanism involving the principal
cell-surface receptor for HA, namely CD44. In addition, HA fragments
and/or CD44 receptor cross-linking have been shown to induce the
expression of the cell adhesion molecules VCAM-1 (14) and
ICAM-1 (15). The CD44 gene codes for a family of alternatively spliced, multifunctional adhesion molecules (reviewed in Ref. 16), which has recently been implicated in the pathogenesis of inflammatory and malignant disease (17, 18, 19). A growing body of evidence is emerging that suggests that, in addition to its function as an adhesion molecule, CD44 also functions as a bioactive signaling receptor. However, a proper understanding of this function of CD44 has been hampered by a lack of insight into the mode in which CD44 communicates with intracellular signal transduction pathways.
The unifying theme in the growing list of genes known to be induced by
HA fragments, via CD44, is that they are all regulated by the
transcription factor NF-B. HA fragments have been shown to activate
NF-B in murine macrophages (20) and increase the
expression of inducible NO synthase (9) and the adhesion
molecule ICAM-1 in an NF-B-dependent manner (15).
NF-B is a dimeric transcription factor that exists in a latent form
in the cytoplasm of unstimulated cells complexed to an inhibitor
protein IB (21, 22). The predominant form of NF-B
activated in cells is a p50/p65 heterodimer that is associated with
IB. Upon stimulation, IB is rapidly phosphorylated on two
critical serine residues (Ser32 and
Ser36), which targets IB for ubiquitination
and subsequent degradation by the 26S proteosome (reviewed in Ref.
22). This allows NF-B to translocate to the nucleus and
activate target genes by binding with high affinity to B elements in
their promoters. The phosphorylation and degradation of IB are
tightly coupled events, and the kinase complex responsible for IB
phosphorylation has recently been identified (23, 24, 25, 26). The
complex comprises two kinases, IB kinase 1 and 2 (IKK-1 and -2),
which form a multimeric protein complex with NF-B essential
modulator (NEMO) (27) and the scaffold protein
IKK-associated protein (IKAP) (28). NEMO and IKAP have
been postulated to function as assembly platforms to facilitate IKK
association with upstream regulators. So far, multiple kinases have
been shown to be upstream activators of the IKK complex.
NF-B-inducing kinase (NIK) is involved in the classical pathway of
NF-B activation by a wide range of stimuli and serves as a point of
convergence of most signals leading to NF-B activation
(29). In addition, the atypical protein kinase C (PKC)
isoform PKC, has also recently emerged as a direct activator of
IKK-2 (30).
To date, the signal transduction mechanisms of CD44-mediated NF-B
activation are unexplored. In this study, we demonstrate the ability of
HA fragments to activate NF-B via CD44 in a diversity of cell lines.
Using T-24 carcinoma cells as a model system, we present evidence that
the pathway activated by HA fragments involves the low molecular weight
G-protein Ras, the atypical PKC isoform PKC, and the IKK complex.
This represents a novel signaling pathway activated by HA fragments
that culminates in NF-B activation, thereby increasing the
expression of NF-B-dependent genes, forming the molecular basis for
the proinflammatory effects of HA fragments.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T-24 human bladder carcinoma, HeLa human cervical carcinoma,
MCF7 breast carcinoma, Jurkat J6.1 human T cell lymphoma, and murine
EL4.NOB-1 thymoma cell lines were obtained from the European Collection
of Animal Cell Cultures (Salisbury, U.K). The murine J774 macrophage
cell line was a gift from Dr. K. Mills (Department of Biology, National
University Maynooth, Kildare, U.K.). Purified HA fragments were
obtained from ICN Biomedicals (Costa Mesa, CA), and, as previously
reported, their peak molecular mass is 
200,000 Da (31).
Native and disaccharide HA was obtained from Sigma (Dorset, U.K.).
Recombinant human IL-1 was a gift from Prof. J. Saklatvala (Kennedy
Institute of Rheumatology, London, U.K.), while TNF- was a gift from
Dr. S. Foster (Zeneca, U.K.). The 22-bp oligonucleotide, 5'-AGT TGA
GGG GAC TTT CCC AGG C-3', containing the NF-B consensus
sequence (underlined) and T4 polynucleotide kinase, was obtained from
Promega (Madison, WI). The 22-bp oligonucleotide, 5'-AGT TGA GGC
GAC TTT CCC AGG C-3', containing the mutated NF-B consensus
sequence (underlined), was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Monoclonal anti-human CD44 standard Ab
(anti-human CD44H) was obtained from R&D Systems (Abingdon, U.K.),
while anti-murine CD44 Ab (KM201) was obtained from Southern
Biotechnology Associates (Birmingham, AL). Monoclonal anti-human
IB was a kind gift from Prof. Ron Hay (University of St. Andrews,
Fife, U.K.). Polyclonal anti-human phospho IB Ab was from New
England Biolabs (Beverly, MA). Antiserum to the NF-B subunits p50,
p65, and c-Rel were a kind gift from Dr. J. Imbert (Institut National
de la Santé et de la Recherche Médicale, Marseille,
France). Polyclonal anti-NEMO was a gift from Drs. S. Yamaoka and
A. Israel (Institute Pasteur, Paris, France). Monoclonal anti-human
CD44 IM7.8.1 was a gift from Dr. D. Buttle (University of Sheffield,
Sheffield, U.K.). Anti-Myc epitope (9E10) Ab was obtained from Upstate
Biotechnology (Lake Placid, NY). Anti-human monoclonal Ras Ab was
obtained from Oncogene Research Products (Cambridge, MA).
[
-32P]ATP (3000 Ci/mmol) was obtained from
Amersham International (Aylesbury, U.K.). Poly(dI · dC) was
obtained from Pharmacia Biosystems (Milton Keynes, U.K.). Calphostin C,
manumycin A, and damnacanthal were obtained from Calbiochem
(Nottingham, U.K.). All other chemicals were obtained from Sigma
(Poole, Dorset, U.K.).
Expression vectors
The pGL3–5B-luc plasmid was a kind gift from Dr. R.
Hofmeister (University of Regensburg, Regensburg, Germany). The pGL1.3
and pGL1.3Bmut plasmids (containing 1344 bp of
the ICAM-1 upstream region (-1353 to -9 relative to the start of
transcription) without or with a mutation at the proximal NF-B site)
were a gift from Dr. K. Catron, (Boehringer Ingelheim Pharmaceuticals,
Ridgefield, CT). Dominant negative expression vectors for IKK-1 and -2
(IKK-1KA and IKK-2KA) were a gift from Dr. D. Goeddel (Tularik, San
Fransisco, CA). pCDNA3- PKCmyc containing
full-length PKC with the myc tag at the COOH terminus and dominant
negative PKC expression vector (PKCmut)
were a gift from Drs. M. Diaz-Meco and J. Moscat (Centro de Biologia
Molecular, Madrid, Spain). The expression vectors encoding amino acids
1–149 of human c-Raf-1 in pGEX-KG (GST-Ras binding domain (RBD)) and
dominant negative N17Ras were obtained from Dr. D. Cantrell (Imperial
Cancer Research Fund, London, U.K.).
Cell culture and treatments
The human T-24 bladder carcinoma cell line was cultured in Medium 199 (HEPES modification), human HeLa cervical carcinoma, and MCF7 breast carcinoma cells were cultured in DMEM medium, while Jurkat T-cell lymphoma, murine EL4.NOB-1 thymoma, and J774 macrophage cell lines were cultured in RPMI 1640 medium. All media contained 10% (v/v) FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Adherant cells (T-24, HeLa, MCF7, and J774) were seeded at 1 x 105ml-1 in six-well plates (3 ml/well) or tissue culture petri dishes (10 ml/dish), while nonadherant cells (Jurkat and EL4.NOB-1) were cultured at 1 x 105ml-1 on day of use. Cells were pretreated with test inhibitors, vehicle control, or left untreated before the addition of stimuli as indicated.
EMSA
Nuclear extracts were prepared as described by Osborn et al.
(32) from confluent T-24, HeLa, MCF7, or J774 cells in
six-well plates (3 ml volume) treated as described in the figure
legends. Jurkat or EL4.NOB-1 cells were collected by centrifugation
before preparation of nuclear extracts as above. In all cases, nuclear
extracts were assessed for NF-B-DNA binding activity, competition,
and supershift analysis as described previously (33).
Immunoblot assay
Confluent T-24, HeLa, MCF7, and J774 cells in six-well plates (3
ml volume) or Jurkat and EL4.NOB-1 cells in 24-well plates (1 ml
volume) were treated as described in the figure legends. For CD44 and
IB analysis, total cell lysate from each well was extracted in
ice-cold radioimmune precipitation buffer (34). Protein
estimations of cell extracts were determined by the dye binding assay
of Bradford (35). For phospho IB analysis,
whole-cell lysates were generated using an SDS sample buffer (62.5 mM
Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v
bromophenol blue). Equal amounts of protein (4–8 µg) in the IB
assay or equal volumes of lysate in the phospho IB assay were
subjected to 10% SDS-PAGE according to the method of Laemmli
(36). Immunoblotting was conducted for CD44, IB, or
phospho IB as previously described (33).
Transient transfection and reporter gene assay
Briefly, confluent T-24 cell monolayers were resuspended after
trypsinization in PBS in 0.4-cm electroporation cuvettes (Invitrogen,
Groningen, The Netherlands). Then, 5 µg of reporter gene plasmid DNA
was added to cells as described in the figure legends. In coexpression
experiments, dominant negative IKK-1 or -2, (IKK-1 or -2KA), PKC
(PKCmut), or Ras (RasN17) were mixed with
reporter gene before electroporation. In all cases, total DNA
transfected (10 µg) was kept constant by supplementation with the
relevant control empty vector (pRK5 for IKK-1 or -2KA, pCDNA3 for
PKCmut, or RSV for RasN17). Then, 48 h
after transfection, cells were treated with HA fragments or other
stimuli for 6 h. Extracts were harvested, and luciferase reporter
gene activity was determined according to the manufacturer’s
recommendations (Promega). The results obtained were normalized for
protein according to the dye binding assay (35).
IKK assay
T-24 cells were seeded in 10-cm dishes (10 ml at 1 x
105/ml for 72 h) and stimulated with HA
fragments (100 µg/ml) as indicated. The IKK complex was
immunoprecipitated using anti-NEMO Ab and assayed using
GST-IB (residues 1–72) as substrate as described previously
(27).
Ras activation assay
T-24 cells were seeded in six-well plates (3 ml at 1 x 105/ml for 48 h). For experiments, cells were cultured in 0.5% FBS for 24 h before stimulation with HA fragments (100 µg/ml) from 1 to 60 min as indicated. Total cell lysates were extracted with lysis buffer (50 mM HEPES, pH 7.4, 10 mM NaF, 10 mM iodoacetamide, 75 mM NaCl, 1% Nonidet P-40, 10 mM MgCl2, 1 mM PMSF, 1 mM NA2VO4, 1 mg/ml ß-glycerol phosphate) for 20 min on ice. Equal amounts of protein per sample were incubated with c-Raf-1 RBD (residues 1–149) (37) precoupled to glutathione agarose beads (50% solution) for 2 h at 4°C. Only activated Ras will bind GST-RBD. Activated protein was then collected by centrifugation at 6000 x g for 5 min, washed four times in lysis buffer, and bound protein eluted by boiling in SDS sample buffer. Samples were subjected to 15% SDS-PAGE according to the method of Laemmli (36). Immunoblotting was conducted for Ras using anti-human monoclonal Ras Ab and visualized by enhanced chemiluminescence.
PKC activation assay
T-24 cells were transfected by electroporation with PKC
containing a myc tag at the COOH terminus
(pCDNA3- PKCmyc). Transfected cells were
cultured in 0.5% FBS before stimulation with HA fragments (100
µg/ml) for 0, 5, or 30 min as indicated. Whole-cell lysates were
extracted and immunoprecipitated with anti-Myc Ab (9E10).
Immune complexes were collected with protein G-Sepharose and
subjected to in vitro kinase assay using 2.5 µg myelin basic protein
(MBP) as substrate as described (38). Samples were
subjected to 15% SDS-PAGE according to the method of Laemmli
(36), and MBP phosphorylation was visualized by
autoradiography.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B by HA fragments
We first investigated the ability of HA fragments to induce
NF-B activation in a variety of human and murine cell lines. Fig. 1
A shows that treatment of
T-24 cells with HA fragments (100 µg/ml for 2 h) at a peak
molecular mass of 200 kDa activated NF-B in T-24 bladder carcinoma
cells, as evidenced by the increased retardation of the DNA probe
containing the B motif (compare lane 2 to lane
1). HA fragments also activated NF-B in HeLa cervical carcinoma
(lane 4), MCF7 breast carcinoma (lane
6) and J774 murine macrophage cells (lane 8). However,
neither EL4.NOB-1 thymoma nor Jurkat T cell lymphoma cells were
responsive to HA fragments (lanes 10 and
13, respectively). The lack of effect of HA fragments in
Jurkat and EL4.NOB-1 cells was not due to a lack of the required
signaling machinery in these cells as activation of NF-B was
observed when cells are treated with IL-1 or TNF-, respectively
(lanes 11 and 14). When cell lines were
probed for expression of standard CD44 with either monoclonal anti-
human (CD44H) or anti-murine (KM201) CD44 Abs, those cell lines
that showed HA activation of NF-B were positive for CD44 expression
as indicated by a band detected at 85 kDa. However, Jurkat and
EL4.NOB-1 cells were negative for CD44 expression (Fig. 1B).
Therefore, this study demonstrates a direct correlation between
expression of CD44 and the ability of HA fragments to activate
NF-B.
|
B. T-24 cells were chosen as the model system for
further studies on HA activation of NF-B as we have previously shown
them to be an excellent model of NF-B activation (33)
and because they are easily transfected. When T-24 cells were
preincubated with an Ab to CD44 (IM7.8.1), which has been shown to
block the binding of HA, HA fragment-induced NF-B activation was
significantly reduced (Fig. 1
C, compare lane 4 to
lane 3). However, IL-1-induced NF-B activation was
unaffected by preincubation with the anti-CD44 Ab (compare
lane 6 to lane 5). Another anti-CD44 mAb
(D2.1) also inhibited the activation of NF-B by HA fragments (data
not shown). These data clearly establish that CD44 plays an important
role in mediating the effects of HA on activation of NF-B.
We next went on to fully characterize the effect of HA fragments in
T-24 cells. Fig. 2A shows a
time course of NF-B activation, the effect being evident from
1 h and peaking at 2 to 8 h. NF-B activity was strong
and sustained and was still detectable after 24 h. The effect of
HA fragments on NF-B activation was also dose-dependent over the
dose range of 1–250 µg/ml as shown in Fig. 2B. Fig. 2C confirms that the protein-DNA complexes activated by HA
fragments were specific for NF-B, because unlabeled NF-B
wild-type consensus sequence dose-dependently competed with binding
activity (compare lanes 3 and 4 to lane
2), while mutant NF-B oligonucleotide containing a single base
pair change in the consensus sequence had no effect (compare lane
6 to lane 2). We also characterized the NF-B
subunits present in the activated complexes by carrying out supershift
analysis. Fig. 2D shows how antisera to both p50 and p65/Rel
A affected the complex (lanes 2 and 3,
respectively), with supershifted bands being detected upon treatment of
extracts with Ab before electrophoresis. There was no detectable
reaction with c-Rel antiserum (lane 4). Hence, HA
fragments activated NF-B complexes containing p50 and p65.
|
B and
stimulate B-linked gene expression in T-24 cells
T-24 cells treated with HA fragments were examined for the
phosphorylation of IB and its subsequent degradation, a critical
event in the activation of this transcription factor. Treatment of
cells with HA fragments induced the phosphorylation of IB on
serine 32, as detected using a polyclonal anti-human IB Ab
that recognizes IB only when phosphorylated on this amino acid
(Fig. 3A). The effect of HA
fragments occurred from 5 min (lane 2) and was
evident up to the last time point examined (60 min; lane 5).
The effect was also dose-dependent, occurring over the concentration
range of 20–150 µg/ml (Fig. 3B). Control levels of
phospho IB were either undetectable (Fig. 3A, lane 1)
or marginal (Fig. 3B, lane 1), depending on whether the
cells were grown in complete medium (Fig. 3B) or in medium
supplemented with 0.5% FBS (Fig. 3A). A marked degradation
of IB protein was also observed (Fig. 3, C and
D). The effect was time-dependent with degradation being
evident at 30 and 60 min (lane 3 and 4)
and dose-dependent from 20 to 200 µg/ml (Fig. 3D).
|
B, cells
were transiently transfected with a B-linked reporter gene, and
reporter gene activity was measured after stimulation with HA
fragments. HA fragments caused a dose-dependent activation of
luciferase reporter gene expression from 10 to 200 µg/ml,
demonstrating that the complexes activated by HA fragments were
transcriptionally active (Fig. 4A). When
transfected cells were preincubated with anti-CD44 mAb, IM7.8.1, HA
fragment-induced B-linked reporter gene activity was inhibited,
confirming the importance of CD44 in mediating the effects of HA
fragments (Fig. 4B). Therefore, the Ab to CD44
blocked both nuclear translocation and transcriptional activity induced
by HA fragments.
|
B site
to demonstrate the involvement of NF-B in HA-induced gene
expression. ICAM-1 reporter gene activity was induced only when the
proximal NF-B consensus site was intact, mutation of the B site
abolishing responsiveness (Fig. 4C). HA fragments
induced reporter gene expression from the ICAM-1 promoter over the dose
range of 10–500 µg/ml (Fig. 4D).
The role of HA size in the activation of NF-B
To address the role of HA size in activation of NF-B, we
investigated the ability of different-sized HA molecules to activate
NF-B, because only low molecular weight HA has been shown to be
active in inducing inflammatory gene expression. As shown in Fig. 5A, treatment of cells with HA
disaccharide (the core subunit of the HA polymer) was incapable of
activating this transcription factor over the dose range of 25–100
µg/ml (lanes 2–4). Similarly, native high
molecular weight HA over the same dose range had no effect on NF-B
activation (lanes 6–8). HA fragments (100
µg/ml) were again active (lane 5). Similarly, when
IB degradation was analyzed, neither native HA (100 µg/ml)
(lane 4) nor HA disaccharide (100 µg/ml)
(lane 2) were capable of inducing IB
degradation, while HA fragments (lane 5) and IL-1
(lane 3) induced degradation. A lack of effect of
native or disaccharide HA on B-linked reporter gene expression was
also observed (Fig. 5C). This data clearly demonstrates how
the size of the HA molecule is an important indicator of
bioactivity.
|
B by HA fragments
We next examined the signal transduction mechanisms involved. We
first investigated the role of IKK-1 and -2. As shown in Fig. 6, A and B, when
T-24 cells were transiently transfected with a B-linked reporter
gene and cotransfected with dominant negative expression vectors
encoding either IKK-1 (Fig. 6A) or -2 (Fig. 6B),
activation of B-linked reporter gene activity by HA fragments was
abolished. Dominant negative IKK-1 and -2 also potently inhibited
reporter gene expression when using IL-1 as a stimulus, a
well-characterized activator of the IKK complex (26). We
next established that HA fragments could activate IKK activity. The IKK
complex was immunoprecipitated with Abs to NEMO (27). Fig. 6C demonstrates that immunoprecipitated complexes showed a
time-dependent increase in phosphorylation of IB-GST (residues
1–72), the substrate for IKK (Fig. 6C, open arrowhead). A
second band of 48 kDa was also phosphorylated in HA fragment-treated
cells (closed arrowhead), which may be NEMO. These data established
that HA fragments were capable of driving IKK activity in T-24 cells
and that such IKK activity was involved in B-linked gene
expression.
|
mediate the activation of NF-B by HA fragments
To elucidate the more proximal events in the pathway to NF-B
activation following ligation of HA fragments with the receptor CD44,
we first focussed our attention on the role that PKC activation plays,
because HA has been shown to activate PKC in cells (39)
and a direct role for PKC in IKK activation has been demonstrated. T-24
cells were pretreated with the PKC inhibitor calphostin C, and the
effect of HA fragments on NF-B activation was determined.
Preincubation of cells with calphostin C dose-dependently inhibited
NF-B activation (Fig. 7A),
with 5 µM abolishing the effect (compare lane 8 to
lane 4). In addition, calphostin C inhibited HA
fragment-induced phosphorylation of IB (Fig. 7B). This implied that activation of PKC in
response to HA was an important step in this signal transduction
cascade. Calphostin C at 5 µM also blocked PMA-induced
phosphorylation of IB, with 5 µM again abolishing the effect
(Fig. 7B, compare lane 11 to lane 9),
demonstrating its efficacy against PKC.
|
has been particularly implicated in
IKK-2 activation (30) and has been shown to be inhibited
by calphostin C (40). Therefore, we focussed our attention
on PKC as the candidate PKC isoform mediating these effects. To do
this, we employed the use of an expression vector encoding a dominant
negative mutant of PKC. The ability of HA fragments to drive
reporter gene expression was inhibited by 5 and 10 µg of plasmid
encoding dominant negative PKC mutant (Fig. 7C). We next examined the effect of HA fragments
on PKC activity. Due to a lack of reliable Abs selective for PKC,
we transfected T-24 cells with a Myc-tagged version of PKC and
examined in vitro kinase activity recovered from immunoprecipitated
PKC before and after stimulation with HA fragments. Fig. 7D demonstrates how treatment of myc PKC-transfected
cells with HA fragments from 5–30 min increased kinase activity as
determined using MBP as substrate (compare lanes 3 and
4 to lane 2). A higher band that migrated at
83 kDa was also observed after prolonged exposures (data not shown),
which may represent autophosphorylated PKC. Immunoblot analysis of
transfected cells using anti-Myc Ab confirmed expression of myc
tagged constructs in transfected cells (data not shown). These
experiments strongly suggest that PKC plays a critical role in
mediating the effects of HA fragments on activation of NF-B.
We next wished to establish the events involved further upstream of
PKC, in particular examining the role that the low molecular weight
G-protein Ras, a known upstream regulator of PKC, may play
(41). First, we used known inhibitors of Ras function to
test whether Ras was involved in the activation of NF-B by HA
fragments. Treatment of T-24 cells with the Ras function inhibitor
damnacanthal (42) dose-dependently inhibited HA fragment
activation of NF-B (Fig. 8A). Damnacanthal from 8
µg/ml abolished the effect (compare lane 7 and
8 to lane 2) and also blocked HA-induced
phoshorylation of IB (Fig. 8B). Another
well-characterized Ras function inhibitor manumycin A
(43), which selectively inhibits protein
farnesyltransferase activity, has also been used to inhibit Ras.
Similarly to damnacanthal, treatment of T-24 cells with manumycin A
showed a dose-dependent inhibition of NF-B activation, with maximal
inhibition occurring from 5 µM (Fig. 8C, lanes 8 and
9). Finally, when T-24 cells were transiently
transfected with a B-linked reporter gene and a vector encoding
dominant negative Ras N17, HA fragment-induced reporter gene activity
was inhibited in cells expressing Ras N17 at 5, 10, and 20 µg of Ras
N17-encoding plasmid (Fig. 8D). Fig. 8E
illustrates the effect of HA fragments on activation of Ras activity as
determined using the Ras-fishing assay, which relies on the ability of
activated Ras to bind to c-Raf-1 (37). Treatment of T-24
cells with HA fragments increased Ras activity from 5 to 60 min (Fig. 8E, lanes 2–6). Taken together, these four mechanistically
distinct approaches establish the importance of Ras in HA signaling to
NF-B.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B
sites, with NF-B playing a critical role in their regulation. In
this study, analysis of CD44 immunoreactivity in a panel of cell lines
establish that NF-B activation by HA fragments directly correlated
with CD44 expression. Further studies in T-24 bladder carcinoma cells
demonstrated that the effect on NF-B, both in terms of DNA binding
and B-linked repoter gene expression, was inhibited by preincubation
with an Ab to CD44 (IM7.8.1). Therefore, a role for CD44 in HA-induced
NF-B activation is clearly indicated. It remains to be determined
whether this phenomenon will also occur in primary cells, but given the
diversity of cell types examined in this study, it seems likely that
these effects will also occur in primary cells.
The activation of NF-B in response to HA fragments was further
characterized in T-24 carcinoma cells. Importantly, HA fragment
activation of NF-B in T-24 cells also occurred in the presence
of the LPS inhibitor, polymyxin B1 (10 µg/ml)
(data not shown). HA fragments induced phosphorylation of IB, an
early event in this pathway as shown by the early time course of
activation, followed by its degradation. Activated complexes, when
assessed for subunit composition, established the presence of both p50
and p65 NF-B subunits. The presence of p65 in the activated complex
suggested that the complex was transcriptionally active.
We also demonstrated that the effects of HA fragments were critically
HA size dependent. High molecular weight HA was inactive, but lower
molecular weight fragments of the size range found at sites of
inflammation were active. In addition, the core subunit of the HA
polymer, HA disaccharide, was inactive. This data is in agreement with
that observed for induction of proinflammatory gene expression by HA
fragments. The basis for the difference between HA fragments and
native HA is unclear. It has been shown that the lack of biological
effect with high molecular weight HA on chemokine expression was not
due to its inability to bind to the cells as it has been shown to bind
to macrophages (31), being displaced by lower molecular
weight forms. This suggests that both higher and lower molecular weight
HA use the same receptor. One possible explanation for the failure of
high molecular weight HA to activate NF-B or induce gene expression
is that the high molecular weight forms may bind to cells in such a way
as to prevent receptor cross-linking. We hypothesize that, in the
context of a noninflammatory milieu, inert native high molecular weight
HA binds "nonproductively" to keep CD44 molecules that are often
highly abundant on cell surfaces in an inactive inert conformation.
Then, lower molecular weight fragments generated under inflammatory
conditions displace higher molecular weight HA and thereby facilitate
CD44 cross-linking, creating a conformation of the receptor that is
biologically active. It is also possible that structural differences
between the different forms of HA, including differences in secondary
or tertiary structure and alterations in the reducing ends of HA, may
contribute to differences in biological activity. However, until more
is known about the physical chemistry of HA fragments generated at
sites of inflammation, it will be difficult to decipher what factors in
addition to fragment molecular weight are important in conferring
biological activity.
Multiple mechanisms have been proposed for the increase in HA turnover
and degradation during inflammatory or malignant disease, leading to
the accumulation of lower molecular weight fragments. Reactive oxygen
species, hyaluronidase activity, and de novo biosynthesis of lower
molecular weight fragments have all been postulated as a mechanism for
the generation of biologically active forms of HA (1, 4, 5). This latter mechanism is of particular interest. Three
distinct human HA synthase enzymes have been identified, namely, HA
synthase 1, 2, and 3 (HAS 1, 2, and 3). HAS 1 and 2 are believed to be
responsible for the synthesis of native high molecular weight HA,
whereas it has been suggested that HAS 3 has a preponderance toward the
biosynthesis of lower molecular weight fragments (44, 45, 46).
Several studies have shown that cytokines and growth factors can
increase HA synthesis in fibroblasts and smooth muscle cells, and
molecular weight analysis shows that the newly synthesized HA is
in the size range that has been found to be biologically active
(5, 47). Indeed, recently it has been demonstrated that
TNF, a key proinflammatory mediator, stimulates the synthesis of
hyaluronan in an NF-B-dependent manner (48), suggesting
that a possible autocrine mechanism exists.
We next examined in detail the signal transduction pathway activated by
HA fragments leading to IB phosphorylation and degradation using
T-24 cells as a model system. Immune complex kinase analysis
demonstrated that IKK complex activity was increased in cells treated
with HA fragments. It is has recently been suggested that the IKKs,
although united in the same heterodimeric complex, activate NF-B
separately and under different conditions (49, 50, 51, 52).
Biochemical and gene knockout studies have suggested that IKK-1 is
essential for NF-B activation in morphogenetic events, including
limb and skeletal pattern formation and proliferation and
differentiation of epidermal keratinocytes (49, 50).
However, IKK-2 may be responsible for NF-B activation by TNF
(51). In our study, transient transfection of expression
vectors encoding either dominant negative IKK-1 or IKK-2 inhibited both
HA- and IL-1-induced reporter gene expression. These effects could be
explained by the fact that overexpression of dominant negative IKK-1
may disrupt the complex and interfere with IKK-2 activity.
Alternatively, the inhibitory effects of dominant negative IKK-1 on HA
fragments may highlight a more interesting phenomenon. The signal(s)
that control IKK-1 activity early in development are unknown, but are
most likely not to be cytokines like IL-1 or TNF. HA and CD44
have long been known to play a role in development, with expression of
CD44 and its variants restricted to certain stages of early limb bud
development (53, 54). The precise role that HA fragments
play in these developmental pathways remains to be determined. Given
that IKK-1 appears to play a role in HA-activated NF-B, it may be
that HA fragment-mediated activation of IKK-1 is important in early
developmental pathways.
Our study also demonstrates a critical role for PKC in the NF-B
pathway, most probably through regulation at the level of IKK activity
(30). Immune complex kinase analysis also demonstrated
that PKC activity was increased in cells treated with HA fragments.
Furthermore, using inhibitors of Ras function and dominant negative
RasN17 we demonstrated a role for the this low molecular weight
G-protein and establish the ability of HA fragments to activate Ras.
Ras is an important upstream regulator of PKC either by a direct
interaction (41) or indirectly through
phosphatidylinositol 3-kinase (55, 56). However, how Ras
is recruited into the signaling pathway following HA binding to CD44 is
unclear. It has been demonstrated that tyrosine kinase activation is a
key early postreceptor signaling event in CD44-mediated signaling
pathways and the role of Ras proteins as downstream effectors of
tyrosine kinase activation pathways is well established
(57). CD44 has been shown to interact with receptor
tyrosine kinases such as ErbB-2, c-Met, and members of the Src
nonreceptor tyrosine kinase family (58, 59). We examined
the role of one of these potential candidates in this process, namely
ErbB-2 (185 kDa), which becomes activated by HA fragments in carcinoma
cells (60). No role for ErbB-2 could be found in the
process of NF-B activation by HA fragments in our studies (data not
shown). Therefore, it remains to be determined what role, if any,
tyrosine phosphorylation may play in HA fragment-induced NF-B
activation and how, following ligand engagement, CD44 can activate
Ras.
In conclusion, we propose a model whereby through CD44, HA fragments
lead to activation of Ras and PKC, followed by activation of the IKK
complex. Therefore, this study provides novel and important insights
into the mechanisms through which HA fragments lead to induction of
proinflammatory genes. The generation of HA fragments at sites of
noninfectious inflammation may prove to be an important mechanism for
the up-regulation of NF-B and thus contribute to the pathological
development of chronic inflammation.
|
Acknowledgments |
|---|
expression
vectors, and Dr. K. Catron (Boehringer Ingelheim Pharmaceuticals,
Ridgefield, CT) for the ICAM-1 promoter constructs. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Luke A. J. O’Neill, Department of Biochemistry and Biotechnology Institute, Trinity College Dublin 2, Ireland. E-mail address:
3 Abbreviations used in this paper: HA, hyaluronic acid; IB, inhibitor protein of NF-B; IKK, IB kinase; NEMO, NF-B essential modulator; IKAP, IKK-associated protein; NIK, NF-B-inducing kinase; PKC, protein kinase C; HAS, HA synthase; RBD, Ras binding domain; MBP, myelin basic protein.
Received for publication May 10, 1999. Accepted for publication December 3, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dependent mechanism in murine macrophages. J. Clin. Invest. 91:2368.
B-dependent mechanism. J. Biol. Chem. 272:8013. in mouse macrophages. J. Immunol. 160:3023. to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages. J. Biol. Chem. 273:35088.B and activating protein-1. J. Immunol. 161:3431.B/I-B autoregulatory loop in murine macrophages. J. Exp. Med. 183:2373.B/rel transcription factor and IB inhibitor system. Curr. Opin. Cell Biol. 5:477.[Medline]
B: a specific inhibitor of the NF-B transcription factor. Science 242:540.B kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
B kinase. Cell 90:373.[Medline]
B kinase-ß: NF-B activation and complex formation with IB kinase- and NIK. Science 278:866.B kinases essential for NF-B activation. Science 278:860.B kinase complex essential for NF-B activation. Cell 93:1231.[Medline]
B kinase complex. Nature 395:292.[Medline]
B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
B kinase ß by protein kinase C isoforms. Mol. Cell Biol. 19:2180. and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor B. Proc. Natl. Acad. Sci. USA 86:2336.B by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304: lack of involvement of H2O2 in NF-B activation by either cytokine in both primary and transformed endothelial cells. J. Biol. Chem. 272:25941.B activation in T lymphocytes: evidence for covalent modification of the p50 subunit. J. Biol. Chem. 270:28557. PKC but not 
/
PKC by caspase-3 during UV- induced apoptosis. J. Biol. Chem. 274:10765. activity: role in 2 integrin and collagenase mRNA expression. J. Cell Biol. 136:473.. J. Biol. Chem. 269:31706. is mediated by the p50/p65 NF-B complex in MRC-5 myofibroblasts. Biochim. Biophys. Acta 1448:416.[Medline]
subunit of IB kinase. Science 284:316.. Science 284:313.B kinase (IKK) is essential for nuclear factor B activation and prevention of apoptosis. J. Exp. Med. 189:1839.B kinase activity through IKKß subunit phosphorylation. Science 284:309. isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 268:13.This article has been cited by other articles:
|
|
 |
|
  Y. Kim, Y.-S. Lee, J. Choe, H. Lee, Y.-M. Kim, and D. Jeoung CD44-Epidermal Growth Factor Receptor Interaction Mediates Hyaluronic Acid-promoted Cell Motility by Activating Protein Kinase C Signaling Involving Akt, Rac1, Phox, Reactive Oxygen Species, Focal Adhesion Kinase, and MMP-2 J. Biol. Chem., August 15, 2008; 283(33): 22513 - 22528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-G. Jiang, Y.-G. Ning, C. Chen, D. Ma, Z.-J. Liu, S. Yang, J. Zhou, X. Xiao, X. A. Zhang, M. L. Edin, et al. Cytochrome P450 Epoxygenase Promotes Human Cancer Metastasis Cancer Res., July 15, 2007; 67(14): 6665 - 6674. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kuribayashi, K. Nakamura, M. Tanaka, T. Sato, J. Kato, K. Sasaki, R. Takimoto, K. Kogawa, T. Terui, T. Takayama, et al. Essential role of protein kinase C {zeta} in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP J. Cell Biol., March 26, 2007; 176(7): 1049 - 1060. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Scheibner, M. A. Lutz, S. Boodoo, M. J. Fenton, J. D. Powell, and M. R. Horton Hyaluronan Fragments Act as an Endogenous Danger Signal by Engaging TLR2 J. Immunol., July 15, 2006; 177(2): 1272 - 1281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ohno, H.-J. Im, C. B. Knudson, and W. Knudson Hyaluronan Oligosaccharides Induce Matrix Metalloproteinase 13 via Transcriptional Activation of NF{kappa}B and p38 MAP Kinase in Articular Chondrocytes J. Biol. Chem., June 30, 2006; 281(26): 17952 - 17960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Firan, S. Dhillon, P. Estess, and M. H. Siegelman Suppressor activity and potency among regulatory T cells is discriminated by functionally active CD44 Blood, January 15, 2006; 107(2): 619 - 627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Takemura, H. Itoh, N. Sagawa, S. Yura, D. Korita, K. Kakui, M. Kawamura, N. Hirota, H. Maeda, and S. Fujii Cyclic mechanical stretch augments hyaluronan production in cultured human uterine cervical fibroblast cells Mol. Hum. Reprod., September 1, 2005; 11(9): 659 - 665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Ducale, S. I. Ward, T. Dechert, and D. R. Yager Regulation of hyaluronan synthase-2 expression in human intestinal mesenchymal cells: mechanisms of interleukin-1{beta}-mediated induction Am J Physiol Gastrointest Liver Physiol, September 1, 2005; 289(3): G462 - G470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-J. Bai, A. P. Spicer, M. M. Mascarenhas, L. Yu, C. D. Ochoa, H. G. Garg, and D. A. Quinn The Role of Hyaluronan Synthase 3 in Ventilator-induced Lung Injury Am. J. Respir. Crit. Care Med., July 1, 2005; 172(1): 92 - 98. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Choi, A. M. Miller, M. J. Nolan, B. Y. J. T. Yue, S. T. Thotz, A. F. Clark, N. Agarwal, and P. A. Knepper Soluble CD44 Is Cytotoxic to Trabecular Meshwork and Retinal Ganglion Cells In Vitro Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 214 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Fitzgerald, D. C. Rowe, B. J. Barnes, D. R. Caffrey, A. Visintin, E. Latz, B. Monks, P. M. Pitha, and D. T. Golenbock LPS-TLR4 Signaling to IRF-3/7 and NF-{kappa}B Involves the Toll Adapters TRAM and TRIF J. Exp. Med., October 6, 2003; 198(7): 1043 - 1055. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. E. Pummill and P. L. DeAngelis Alteration of Polysaccharide Size Distribution of a Vertebrate Hyaluronan Synthase by Mutation J. Biol. Chem., May 23, 2003; 278(22): 19808 - 19814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Slevin, S. Kumar, and J. Gaffney Angiogenic Oligosaccharides of Hyaluronan Induce Multiple Signaling Pathways Affecting Vascular Endothelial Cell Mitogenic and Wound Healing Responses J. Biol. Chem., October 18, 2002; 277(43): 41046 - 41059. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ghatak, S. Misra, and B. P. Toole Hyaluronan Oligosaccharides Inhibit Anchorage-independent Growth of Tumor Cells by Suppressing the Phosphoinositide 3-Kinase/Akt Cell Survival Pathway J. Biol. Chem., October 4, 2002; 277(41): 38013 - 38020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. F. Weber, R. T. Bronson, J. Ilagan, H. Cantor, R. Schmits, and T. W. Mak Absence of the CD44 Gene Prevents Sarcoma Metastasis Cancer Res., April 1, 2002; 62(8): 2281 - 2286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. P. Toole Hyaluronan promotes the malignant phenotype Glycobiology, March 1, 2002; 12(3): 37R - 42R. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Turley, P. W. Noble, and L. Y. W. Bourguignon Signaling Properties of Hyaluronan Receptors J. Biol. Chem., February 8, 2002; 277(7): 4589 - 4592. [Full Text] [PDF]
|
|
|
|
|
|
D. J. Mahoney, R. T. Aplin, A. Calabro, V. C. Hascall, and A. J. Day Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length Glycobiology, December 1, 2001; 11(12): 1025 - 1033. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. SCURI, W. M. ABRAHAM, Y. BOTVINNIKOVA, and R. FORTEZA Hyaluronic Acid Blocks Porcine Pancreatic Elastase (PPE)-induced Bronchoconstriction in Sheep Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): 1855 - 1859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Sohara, N. Ishiguro, K. Machida, H. Kurata, A. A. Thant, T. Senga, S. Matsuda, K. Kimata, H. Iwata, and M. Hamaguchi Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen-activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner Mol. Biol. Cell, June 1, 2001; 12(6): 1859 - 1868. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wang, Y. Zhan, L. Xu, G. Z. Feuerstein, and X. Wang Use of Suppression Subtractive Hybridization for Differential Gene Expression in Stroke : Discovery of CD44 Gene Expression and Localization in Permanent Focal Stroke in Rats Stroke, April 1, 2001; 32(4): 1020 - 1027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fujimoto, H. Kawashima, T. Tanaka, M. Hirose, N. Toyama-Sorimachi, Y. Matsuzawa, and M. Miyasaka CD44 binds a chondroitin sulfate proteoglycan, aggrecan Int. Immunol., March 1, 2001; 13(3): 359 - 366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Diaz-Meco and J. Moscat MEK5, a New Target of the Atypical Protein Kinase C Isoforms in Mitogenic Signaling Mol. Cell. Biol., February 15, 2001; 21(4): 1218 - 1227. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. D. Camenisch and J. A. McDonald Hyaluronan . Is Bigger Better? Am. J. Respir. Cell Mol. Biol., October 1, 2000; 23(4): 431 - 433. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or incubated with HA
fragments 150 µg/ml (
) for 6 h. Cell lysates were prepared
and analyzed for luciferase reporter gene activity and normalized for
protein concentration. Results represent fold-stimulation over
control ± SD for a single experiment (performed in triplicate)
and is representative of three experiments. D, T-24
cells were transiently transfected by electroporation with Myc-tagged
PKC