From the
Gonda Department of Cell and Molecular
Biology, House Ear Institute, and the Department of Otolaryngology, University
of Southern California, Los Angeles, California 90057, the
Department of Molecular Medicine, Graduate
School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973,
Japan, the ¶Gastrointestinal Research Laboratory,
Veterans Affairs Medical Center and Department of Medicine, University of
California, San Francisco, California 94143, and the
||Michael E. DeBakey Department of Surgery and
Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, Texas 77030
Received for publication, February 19, 2003 , and in revised form, May 3, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mucins, the major component of mucous secretions, are high molecular weight
and heavily glycosylated proteins synthesized by the mucosal epithelial cells
lining the middle ear, trachea, and digestive and reproductive tracts
(1217).
They protect the epithelial surface by binding and trapping inhaled infectious
particles, including bacteria and viruses, for mucociliary clearance, at least
in part because of the extraordinary diversity of their carbohydrate side
chains (13,
14). Therefore, up-regulation
of mucin in infectious diseases represents an important host innate defensive
response to microbes (13).
However, in patients with otitis media with effusion and chronic obstructive
pulmonary diseases whose mucociliary clearance mechanisms have become
defective, excessive production of mucin will lead to airway obstruction in
chronic obstructive pulmonary disease and conductive hearing loss in otitis
media with effusion
(1824).
To date, 18 mucin genes have been identified
(1417).
Among these, at least MUC2, MUC5AC, and MUC5B have been
shown to play an important role in the pathogenesis of respiratory infectious
diseases (14,
15,
1721).
We have demonstrated recently that TGF--Smad signaling pathway
cooperates with NF-
B to mediate nontypeable Haemophilus
influenzae-induced MUC2 mucin transcription
(24). Still unknown is whether
or not the TGF-
-Smad signaling pathway regulates MUC5AC,
another key member of the mucin superfamily, in a similar manner.
Understanding how TGF-
signaling mediates up-regulation of
MUC5AC mucin may not only bring new insights into the novel role of
TGF-
signaling in attenuating host primary innate defensive responses
and but may also open up novel therapeutic targets for these diseases.
In addition to the TGF--Smad signaling pathway, p38 MAPK, consisting
of four isoforms,
,
,
and
, together with its
upstream MAPK kinases (MKK3/6), comprises another important signaling pathway
mediating diverse cellular responses
(25). Recent studies have
shown that p38 MAPK is not only regulated by its immediate upstream kinases
such as MKK3/6 but is also regulated by other signaling pathways such as
TGF-
signaling pathway
(26,
27). In contrast to the
relatively extensive studies on the positive regulation of p38 by TGF-
signaling, relatively little is known about the negative regulation of p38
MAPK by TGF-
signaling, especially in the pathogenesis of bacterial
infectious diseases.
Because of the important role of TGF- signaling in mediating diverse
cellular responses and our recent observations showing the activation of
TGF-
and p38 signaling by nontypeable H. influenzae (NTHi) as
well as the reported interaction between TGF-
and p38 pathways, we
hypothesized that the TGF-
-Smad signaling pathway interacts with p38 to
mediate up-regulation of MUC5AC mucin in response to NTHi in human
epithelial cells. Here, we showed that NTHi, a major human bacterial pathogen
of otitis media and chronic obstructive pulmonary disease, strongly induces
up-regulation of MUC5AC mucin
(2833),
a primary innate defensive response for mammalian airways, via activation of
multiple signaling pathways. The activation of the TLR2-MyD88-dependent p38
MAPK pathway is required for NTHi-induced MUC5AC transcription,
whereas activation of TGF-
receptor-mediated signaling leads to
down-regulation of p38 MAPK by inducing MAPK phophatase-1 (MKP-1) expression,
thereby acting as a negative regulator for MUC5AC induction. These
studies bring new insights into the novel role of TGF-
signaling in
attenuating host primary innate defensive responses and enhance our
understanding of the negative cross-talks between the TGF-
-Smad and p38
MAPK signaling pathways.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial Strains and Culture ConditionsNTHi strain 12, a clinical isolate, was used in this study (21, 24, 33). Bacteria were grown on chocolate agar at 37 °C in an atmosphere of 5% CO2. For making NTHi crude extract, NTHi were harvested from a plate of chocolate agar after overnight incubation and incubated in 30 ml of brain heart infusion broth supplemented with NAD (3.5 µg/ml). After overnight incubation, NTHi were centrifuged at 10, 000 x g for 10 min, and the supernatant was discarded. The resulting pellet of NTHi was suspended in 10 ml of phosphate-buffered saline and sonicated. Subsequently, the lysate was collected and stored at 70 °C. NTHi lysates (5 µg/ml) were used in all the experiments. We chose to use NTHi lysates because of the following reasons. First, NTHi has been shown to be highly fragile and has the tendency to autolyze. Its autolysis can be triggered in vivo under various conditions including antibiotic treatment (21, 24, 33). Therefore, using lysates of NTHi represents a common clinical condition in vivo, especially after antibiotic treatment.
Cell CultureHuman colon epithelial cell line HM3 was
maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (Invitrogen) (21,
24). Human cervix epithelial
cell line HeLa and human middle ear epithelial cell line HMEEC-1 were
maintained as described (17,
28). Primary human airway
epithelial (NHBE) cells were purchased from Clonetics (San Diego, CA). NHBE
cells were maintained in Clonetics' recommended bronchial epithelial growth
medium, which includes supplements of bovine pituitary extract,
hydro-cortisone, human recombinant epidermal growth factor, epinephrine,
transferrin, insulin, retinoic acid, triiodothyronine, gentamicin, and
amphotericin B (Clonetics), to a confluence of 6080% (37 °C, 5%
CO2). The media were replaced every other day. Only cells of
passage 4 were used for experiments. Wild type mink Mv1Lu cells and mutant
cell lines, DR26 that are derived from Mv1Lu and lack functional TGF-
receptor type II, were kindly provided by Dr. Joan Massague (Memorial
Sloan-Kettering Cancer Center, New York), and were maintained as described
previously (24,
34). All media received
additions of 100 units/ml penicillin and 0.1 mg/ml streptomycin.
Reverse Transcription-PCR Analysis of MKP-1Total RNA was isolated from human epithelial cells using a Qiagen kit (Valencia, CA) following the manufacturer's instruction. For the reverse transcription reaction, the Moloney murine leukemia virus preamplification system (Invitrogen) was used. PCR amplification was performed with Taq gold polymerase (PerkinElmer Life Sciences). The oligonucleotide primers were: MKP-1, 5'-GCT GTG CAG CAA ACA GTC GA-3' and 5'-CGA TTA GTC CTC ATA AGG TA-3', and cyclophilin, 5'-CCG TGT TCT TCG ACA TTG CC-3' and 5'-ACA CCA CAT GCT TGC CAT CC-3'.
Real Time Quantitative Reverse Transcription-PCR AnalysisTotal RNA was isolated from human epithelial cells using TRIzol® Reagent (Invitrogen) following the manufacturer's instruction. For the real time quantitative reverse transcription-PCR, Pre-Developed TaqMan Assay Reagents (Applied Biosystems) were used. Synthesis of cDNA from total RNA samples was performed with MultiScribeTM Reverse Transcriptase. To normalize MUC5AC expression relative to cDNA, we used primers and a TaqMan probe corresponding to cyclophilin. Expression of MUC5AC was measured relative to cyclophilin. Primers and the TaqMan probe for MUC5AC were designed by using Primer Express software (Applied Biosystems) and synthesized by Applied Biosystem Customer Oligo Synthesis Service (Applied Biosystems). TaqMan probes were labeled with 6-carboxyfluorescein on the 5' end and 6-carboxytetramethylrhodamine on the 3' end. The primers and probes for MUC5AC were: forward primer, 5'-GTTCTATGAGGGCTGCGTCTTT-3'; reverse primer, 5'-GGCTGGAGCACACCACATC-3'; and TaqMan probe, 5'-FAM-ACCGGTGCCACATGACGGACCT-TAMARA-3'. The reactions were amplified and quantified using an ABI 7700 sequence detector and the manufacturer's software (Applied Biosystems). The relative quantity of MUC5AC mRNA was obtained using the comparative CT method (for details, see user Bulletin 2 for the ABI PRISM 7700 Sequence Detection System under www.appliedbiosystems.com/support/tutorials) and was normalized using Pre-Developed TaqMan Assay Reagent Human Cyclophilin as an endogenous control (Applied Biosystems).
Plasmids, Transfections, and Luciferase AssaysThe
expression plasmids fp38(AF), fp38
(AF), MyD88 DN, hTLR2 DN and
wild type, T
RII DN and wild type, Smad3 DN and wild type, Smad4 DN and
wild type were previously described
(24,
33,
35). The expression plasmids
of the wild type and antisense MKP-1 were kindly provided by Dr. N. Tonks
(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Dr. C.
Desbois-Mouthon (INSERM U-402, Faculté de Médecine
Saint-Antoine, Paris, France), respectively
(36). The reporter construct,
human MUC5AC regulatory region, was subcloned into upstream of a
TK-32 promoter luciferase
vector.2 SBE-luc was
previously described (24,
37). All of the transient
transfections were carried out in triplicate using TransIT-LT1 reagent (Mirus,
Madison, WI) following the manufacturer's instructions, unless otherwise
indicated. In all co-transfections, an empty vector was used as a control. The
transfected cells were pretreated with or without chemical inhibitors
including SB203580, cycloheximide, and Ro-31-8220 for 1 h. NTHi or recombinant
TGF-
1 was then added to the transfected cells 42 h after transfection.
After 5 h, the cells were harvested for luciferase assay. In experiments using
neutralization TGF-
antibody, NTHi lysates were pretreated with either
TGF-
neutralization antibody or control antibody for 1 h before being
added to the transfected cells for 5 h.
Western Blot AnalysisIn all transfections with either a
wild type or a dominant-negative mutant of signaling molecules, an empty
vector was used as a control. Transfected cells were treated with or without
NTHi 42 h after transfection. Antibodies against phospho-p38 (Thr-180/182) and
p38 were purchased from Cell Signaling (Beverly, MA). Antibodies against
phospho-TRII (Tyr-336) and T
RII were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Phosphorylation of p38 and T
RII
were detected as described and following the manufacturer's instructions
(24,
33).
Immunofluorescent StainingThe cells were cultured on
four-chamber microscope slides. After NTHi or TGF-1 treatment, the cells
were fixed in paraformaldehyde solution (4%) and incubated with mouse
anti-Smad4 monoclonal antibody for 1 h (Santa Cruz Biotechnology, Inc.).
Primary antibody was detected with fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (Santa Cruz Biotechnology, Inc.). The samples were viewed and
photographed using a Zeiss Axiophot microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We next sought to determine the receptor-mediated signaling pathway
upstream of p38 that mediates NTHi-induced MUC5AC transcription.
Based on our recent finding that NTHi induces p38-dependent activation of
NF-B via TLR2 (24,
33), we therefore determined
the involvement of TLR2 signaling in MUC5AC induction. Interestingly,
co-transfection of HM3 cells with a dominant-negative mutant of TLR2 inhibited
NTHi-induced MUC5AC transcription, whereas overexpression of a wild
type TLR2 enhanced MUC5AC induction
(Fig. 2A). Similarly,
overexpressing a dominant-negative mutant MyD88, a key adapter protein
downstream of TLR2 (38), also
inhibited MUC5AC induction. Concomitantly, overexpression of
dominant-negative mutants of TLR2 and MyD88 also abrogated MUC5AC
induction at the mRNA level as assessed by performing real time quantitative
PCR analysis (Fig.
2B). To address whether the TLR2-MyD88 signaling pathway
also mediates NTHi-induced MUC5AC in primary human bronchial
epithelial cells, we next studied the effects of overexpressing
dominant-negative mutants of TLR2 and MyD88 on MUC5AC induction in
NHBE cells. As shown in Fig.
2C, NTHi-induced MUC5AC expression at mRNA level
was also abolished by these treatments in primary NHBE cells. These data
indicate that the TLR2-MyD88 signaling pathway is required for MUC5AC
induction.
|
To further determine whether TLR2-MyD88 acts upstream of p38 MAPK, we assessed the effect of overexpressing the same dominant-negative mutants of TLR2 and MyD88 on NTHi-induced p38 phosphorylation by using anti-phosphorylated p38 MAPK antibody. Indeed, co-transfecting the HM3 cells with both dominant-negative mutants inhibited NTHi-induced p38 phosphorylation (Fig. 2D). Thus, it is evident that the TLR2-MyD88 signaling pathway mediates NTHi-induced up-regulation of MUC5AC via activation of p38 MAPK.
TGF- Type II Receptor-Smad3/4 Signaling
Negatively Regulates NTHi-induced MUC5AC TranscriptionBecause of
the important role TGF-
signaling plays in regulating host immune
responses in bacterial infections and our recent study showing the positive
involvement of TGF-
signaling in NTHi-induced transcription of
MUC2 (8,
24), another key member of
mucin superfamily, we were interested in determining whether
TGF-
-receptor signaling also mediates NTHi-induced up-regulation of
MUC5AC mucin, a primary innate defensive response for host
respiratory mucosa (13). We
first examined the effects of overexpressing the dominant-negative mutant and
wild type of T
RII on NTHi-induced MUC5AC expression
(40,
41). Surprisingly,
co-transfecting the HM3 cells with a dominant-negative mutant of T
RII
greatly enhanced NTHi-induced MUC5AC expression at the mRNA level,
whereas overexpressing the wild type T
RII attenuated MUC5AC
induction (Fig. 3A).
We next confirmed the negative involvement of T
RII signaling in
MUC5AC induction in primary human bronchial epithelial NHBE cells. As
shown in Fig. 3B,
co-transfecting NHBE cells with a dominant-negative T
RII also enhanced
MUC5AC induction at the endogenous mRNA level. Thus, these data
indicate that T
RII signaling negatively regulates NTHi-induced
MUC5AC expression in human epithelial cells.
|
To further confirm whether TRII signaling indeed acts as a negative
regulator for MUC5AC induction, we took advantage of the available
DR26 cell, a lung epithelial cell line that is derived from the wild type
Mv1Lu cells and that lacks functional T
RII
(24,
34). We first assessed the
effect of TGF-
1 on SBE-dependent promoter activity in Mv1Lu and DR26
cells, respectively. As expected, no TGF-
1-induced promoter activity was
observed in mutant DR26 cells, whereas the wild type Mv1Lu cells showed potent
induction of SBE-dependent promoter activity by TGF-
1
(Fig. 3C, left
panel). We next examined the effects of NTHi on MUC5AC promoter
activity in the same cells, respectively. As shown in
Fig. 3C (right
panel), almost no MUC5AC induction by NTHi was observed in wild
type Mv1Lu cells, whereas NTHi induced MUC5AC promoter activity in
mutant DR26 cells by
2.5-fold. In accordance with these results,
co-transfecting the wild type Mv1Lu cells with a dominant-negative mutant
T
RII abolished the TGF-
1-induced SBE response, whereas
co-transfecting the mutant DR26 cells with a wild type T
RII rescued the
SBE response to TGF-
1 (Fig.
3D, upper panels). In contrast to the SBE
response to TGF-
1, overexpressing a dominant-negative mutant T
RII
in wild type Mv1Lu cells rendered the cells MUC5AC-responsive to
NTHi, whereas co-transfecting the mutant DR26 cells with a wild type
T
RII abolished the MUC5AC response to NTHi
(Fig. 3D, lower
panels). Thus, these data suggest that T
R signaling is a negative
regulator for NTHi-induced MUC5AC mucin transcription in human
epithelial cells.
Because the negative involvement of TR signaling in MUC5AC
induction was determined mainly by using overexpression of T
R expression
plasmids and the T
R mutant cell lines, we next sought to confirm whether
NTHi indeed activates TGF-
signaling like TGF-
does. We first
evaluated the effect of NTHi on phosphorylation of T
RII by using an
antibody against phosphorylated T
RII. As shown in
Fig. 3E (left
panel), NTHi, like TGF-
1, induced phosphorylation of T
RII. We
next investigated whether NTHi activates TGF-
-Smad signaling by
evaluating NTHi-induced nuclear translocation of Smad4, a key step for the
Smad3/4 complex to exert its transcriptional activity
(2).
Fig. 3E (right
panel) shows that NTHi potently induces nuclear translocation of Smad4.
As expected, Smad4 translocation was also induced by TGF-
1. To further
confirm whether NTHi activates TGF-
-Smad-dependent transcriptional
activity, we assessed the effect of NTHi on SBE-dependent promoter activity by
using SBE luciferase reporter in HM3 cells
(45). When we exposed the
transfected cells to NTHi or TGF-
1, SBE-driven luciferase activity
increased in cells treated with NTHi or TGF-
1
(Fig. 3E, right
panel). Taken together, these results confirm that NTHi, like
TGF-
1, indeed activates TGF-
-Smad signaling pathway, which in turn
leads to the inhibition of NTHi-induced MUC5AC transcription.
Although we have demonstrated that TGF- signaling acts as a negative
regulator for NTHi-induced MUC5AC transcription, it is still unclear
how TGF-
receptor-mediated signaling is activated by NTHi in the
regulation of MUC5AC transcription. Based on our recent findings that
NTHi did not induce any detectable increase in three major TGF-
family
members, TGF-
1, 2, and 3 in the conditioned medium of HM3 cells and that
the early NTHi-induced T
RII phosphorylation was observed at 5 min upon
treatment (24), it is likely
that NTHi may activate TGF-
-Smad signaling via a TGF-
autocrine-independent mechanism. To determine whether NTHi-derived
TGF-
-like factor is responsible for the negative regulation of
NTHi-induced MUC5AC transcription mediated by TGF-
signaling,
we assessed the effect of NTHi lysates pretreated with TGF-
neutralization antibody or control antibody on the transcriptional activity of
MUC5AC promoter. As shown in Fig.
3F, NTHi-induced MUC5AC promoter activity was
enhanced by TGF-
neutralization antibody treatment, whereas NTHi-induced
MUC5AC transcription remained unchanged upon treatment with control
antibody. Collectively, these data suggest that TGF-
receptor-mediated
signaling is likely activated by a NTHi-derived TGF-
-like factor via a
mechanism independent of TGF-
1, 2, and 3 autocrine signaling in the
negative regulation of NTHi-induced MUC5AC transcription. It should
be noted that our data do not preclude the involvement of the latent
TGF-
s stored in the extracellular matrix that might be activated by NTHi
and then cross-talk with T
R. In addition, it is still unclear whether
other TGF-
family members may be involved in mediating the negative
regulation of NTHi-induced MUC5AC transcription in an
autocrine-dependent manner.
Because of the importance of Smads in transducing TGF-
receptor-mediated signals into the nucleus
(5,
37,
39), we next sought to
determine the involvement of Smad3, one of the key receptor-activated Smads,
and Smad4, the Co-Smad (common partner Smad). As shown in
Fig. 4A,
overexpression of a dominant-negative mutant of either Smad3 or Smad4 enhanced
NTHi-induced MUC5AC expression, whereas co-transfection with the wild
type form of either Smad3 or Smad4 inhibited MUC5AC induction at the
mRNA level as assessed by performing real time quantitative PCR analysis in
HM3 cells. In addition, activation of TGF-
-Smad signaling by
co-expression of wild type Smad3 and Smad4 markedly abrogated MUC5AC
induction at the mRNA level (Fig.
4B). Similar to their inhibitory effect on
MUC5AC induction at the mRNA level, co-expression of wild type Smad3
and Smad4 also abolished NTHi-induced MUC5AC at the transcriptional
level as evaluated by co-transfecting the HM3 cells with a MUC5AC
promoter luciferase reporter construct
(Fig. 4C).
Collectively, these results demonstrated that TGF-
type II
receptor-Smad3/4 signaling is negatively involved in NTHi-induced
MUC5AC transcription, thereby inhibiting the primary innate defensive
response in host airway mucosa.
|
TRII-Smad3/4 Signaling Pathway Negatively
Mediates NTHi-induced MUC5AC via a Negative Cross-talk with p38
MAPKHaving identified TLR2-MyD88-p38 MAPK as a positive pathway
and T
R-Smad3/4 as a negative pathway involved in NTHi-induced
MUC5AC transcription, it is still unknown whether or not there is a
negative cross-talk between these two signaling pathways. To test the
hypothesis that TGF-
signaling negatively regulates NTHi-induced
MUC5AC transcription via down-regulating the p38 MAPK activity, we
first investigated the effect of overexpressing a dominant-negative mutant of
T
RII on NTHi-induced p38 phosphorylation. As shown in
Fig. 5A, NTHi-induced
p38 phosphorylation was greatly enhanced in HM3 cells transfected with a
dominant-negative mutant of T
RII. The enhanced p38 phosphorylation was
observed in HM3 cells treated with NTHi for various times. The highest level
of the enhanced p38 phosphorylation was observed at 60 min. In addition, the
enhancement of NTHi-induced p38 phosphorylation by inhibition of TGF-
signaling were also observed in primary bronchial epithelial NHBE cells
(Fig. 5B). Similarly,
co-transfecting the HM3 cells with a dominant-negative mutant of either a
Smad3 or Smad4 also markedly enhanced the NTHi-induced p38 phosphorylation
(Fig. 5C). In
agreement with these results, addition of exogenous TGF-
1 in parallel
with NTHi attenuated NTHi-induced p38 phosphorylation in both HM3 and primary
NHBE cells (Fig. 5, D and
E). Moreover, exogenous TGF-
1 also inhibited
NTHi-induced MUC5AC transcription in a dose-dependent manner
(Fig. 5F). These
results thus confirmed our hypothesis that T
R-Smad3/4 signaling indeed
acts as a negative regulator for NTHi-induced MUC5AC transcription
via inhibiting p38 activation.
|
TGF--Smad Signaling Negatively Regulates NTHi-induced
MUC5AC Induction via a MAPK Phosphatase-1-dependent Inhibition of p38
MAPKOne key issue that has yet to be addressed is how
TGF-
-Smad signaling down-regulates NTHi-induced p38 activation. We first
determined the possible involvement of de novo protein synthesis in
NTHi-induced MUC5AC expression at the mRNA level by using the protein
synthesis inhibitor cycloheximide. As shown in
Fig. 6A, NTHi-induced
up-regulation of MUC5AC at the mRNA level was further enhanced in HM3
cells pretreated with cycloheximide, indicating that de novo protein
synthesis is negatively involved NTHi-induced MUC5AC expression.
Based on this finding and the evidence that T
R signaling down-regulates
NTHi-induced p38 phosphorylation, it is logical that T
R-Smad3/4
signaling may be involved in up-regulation of an inhibitor for p38 MAPK. In
review of the known inhibitors for p38, MKP-1, a member of a novel class of
dual specificity phosphatases collectively termed MAPK phosphatases,
represents a key protein phosphatase that dephosphorylates and inactivates p38
MAPK (36,
42). To determine whether
T
R-Smad3/4 signaling is involved in the NTHi-induced MKP-1 expression,
we next evaluated the effect of overexpression of a dominant-negative mutant
of T
RII on NTHi-induced MKP-1 expression at the mRNA level.
Interestingly, NTHi greatly induced MKP-1 expression at the mRNA level in a
time-dependent manner, and the MKP-1 induction was greatly inhibited by
overexpressing a dominant-negative mutant of T
RII
(Fig. 6B). A similar
result was also observed in primary bronchial epithelial NHBE cells (data not
shown). Therefore, T
R signaling appears to be involved in NTHi-induced
MKP-1 expression, which in turn leads to down-regulation of p38-dependent
MUC5AC transcription.
|
To confirm the negative involvement of MKP-1 in NTHi-induced
MUC5AC up-regulation, we then assessed the effect of Ro-31-8220, a
chemical inhibitor for MKP-1 expression
(36), on MUC5AC
induction by NTHi in HM3 cells. As expected, Ro-31-8220 indeed enhanced
NTHi-induced up-regulation of MUC5AC in a dose-dependent manner
(Fig. 5C). To further
confirm whether MKP-1 is indeed negatively involved in MUC5AC
induction, we next investigated the effects of overexpressing an antisense and
a wild type full-length MKP-1 construct on NTHi-induced MUC5AC
expression (36,
42,
43). As shown in
Fig. 6D,
overexpression of the antisense MKP-1 construct enhanced MUC5AC
induction, whereas co-expression of a wild type MKP-1 attenuated
MUC5AC induction. Taken together, our data demonstrated that
TGF--Smad signaling is negatively involved in NTHi-induced
MUC5AC induction via MKP-1-dependent inhibition of p38 MAPK.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
A major finding in this study is the experimental evidence for the negative
regulation of host mucosal defensive response to bacterial pathogen by
TGF- signaling. In review of the role of TGF-
signaling in
infectious diseases (8), most
studies have focused on pathogens that infect host macrophages such as T.
cruzi and a variety of Leishmania species. These studies have
demonstrated that excessively produced TGF-
upon infection inhibits
macrophage activation, thereby favoring virulence
(911).
In certain situations, however, there is also evidence that TGF-
has
been correlated with enhanced resistance to microbes such as C.
albicans (12), thus
benefiting the host. Despite these distinct observations that mainly focused
on macrophages, little is known about how TGF-
regulates host innate
defensive responses, such as up-regulation of mucin
(13), in the mucosal
epithelial cells of airway. Therefore, our study may bring new insights into
the novel role of TGF-
signaling in attenuating host primary innate
defensive responses to respiratory bacterial pathogens and may open up novel
therapeutic targets for treatment of airway infectious diseases.
Another interesting finding in this study is the negative cross-talk
between the TGF--Smad3/4 signaling pathway and the p38 MAPK pathway.
Experimental evidence over the past few years has suggested that
TGF-
-Smad pathway may signal through interactions with other signaling
pathways such as p38 MAPK. Although most of these studies have demonstrated an
important role of TGF-
signaling in activation of p38 MAPK
(26,
27), the negative regulation
of p38 MAPK by TGF-
-Smad signaling still remains largely unknown. In the
present study, we provided first hand evidence that activation of
TGF-
-Smad3/4 signaling by bacterium NTHi leads to attenuation of p38
MAPK via up-regulation of MKP-1 in human airway epithelial cells. These
observations should further enhance our understanding of the signaling
mechanisms underlying the functional cross-talk between the TGF-
-Smad
signaling pathway and the p38 MAPK pathway.
Finally, interesting evidence was also provided for the possible
involvement of NTHi-derived TGF--like factor in activation of the
TGF-
-Smad signaling pathway, which in turn leads to the negative
regulation of NTHi-induced MUC5AC transcription. Several lines of
evidence support this notion. First, the NTHi-induced T
RII
phosphorylation was observed at as early as 5 min
(24). Given such an early
phosphorylation of T
RII, it is likely that the early phosphorylation of
T
RII may occur as a result of direct activation of T
R signaling by
NTHi rather than NTHi-induced TGF-
autocrine signaling. Although
T
RII is generally known as a serine/threonine kinase, there is also
evidence that T
RII can function as a dual specificity kinase, and
tyrosine phosphorylation may have an important role in T
R signaling
(44). Thus, NTHi-induced
tyrosine phosphorylation of T
RII at 5 min may be at least interpreted as
a T
R-mediated early response to NTHi factors. Second, pretreatment of
NTHi lysates with the TGF-
neutralization antibody enhanced its ability
to induce the transcriptional activity of MUC5AC promoter as compared
with the NTHi lysates treated with control antibody. Finally, NTHi did not
induce any detectable increase in three major TGF-
family members,
TGF-
1, 2, and 3, in the conditioned medium of HM3 cells. Taken together,
these data suggest that TGF-
-Smad signaling pathway is likely activated
by NTHi-derived TGF-
-like factor via a mechanism independent of
TGF-
1, 2, and 3 autocrine signaling in the negative regulation of
NTHi-induced MUC5AC transcription. However, our data do not rule out
the possible involvement of the latent TGF-
s stored in the extracellular
matrix that might be activated by NTHi and then cross-talk with T
RII. In
addition, it is still unclear whether other TGF-
family members are
involved in mediating the negative regulation of NTHi-induced MUC5AC
transcription in an autocrine-dependent manner. Future studies will focus on
determining the molecular identity of NTHi-derived TGF-
-like factors and
whether NTHi also activates the latent TGF-
stored in the extracellular
matrix that in turn leads to the activation of TGF-
signaling. In
addition, how TGF-
signaling up-regulates MKP-1 expression via a
Smad3/4-dependent mechanism will be further explored using biochemical and
genetic approaches. Finally, the role of MKP-1 in mediating the negative-cross
talk between TGF-
-Smad pathway and the p38 pathway in vivo will
also be confirmed by using MKP-1 knockout mice. These studies should deepen
our understanding of the role of TGF-
signaling in regulating host
innate defensive response to respiratory bacterial pathogens.
![]() |
FOOTNOTES |
---|
** To whom correspondence should be addressed: House Ear Inst., 2100 West Third St., Los Angeles, CA 90057. E-mail: jdli{at}hei.org.
1 The abbreviations used are: TGF-, transforming growth factor-
;
MAPK, mitogen-activated protein kinase; NTHi, nontypeable H.
influenzae; MKP-1, MAPK phophatase-1; DN, dominant-negative; TLR2,
Toll-like receptor 2; T
RII, TGF-
receptor type II; SBE,
Smad-binding element.
2 H. Jono, H. Xu, H. Kai, D. J. Lim, Y. S. Kim, X.-H. Feng, and J.-D. Li,
unpublished data.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|