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J. Biol. Chem., Vol. 280, Issue 52, 42766-42773, December 30, 2005
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B Kinase and IB
Reveal NF-B-dependent as Well as NF-B-independent Pathways of HAS1 Activation*
From the Ludwig Boltzmann Institute for Rheumatology and Balneology, 1100 Vienna, Austria
Received for publication, March 28, 2005 , and in revised form, August 2, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B in induced and noninduced HAS activation. Transforming growth factor (TGF) 
1 as well as interleukin (IL)-1 are both strong inducers of HAS1 transcription. Stimulation of fibroblast-like synoviocytes with IL-1 resulted in rapid degradation of IB
, an event that was preceded by IB phosphorylation. Interestingly, TGF1 neither affected IB levels, nor did it cause phosphorylation of IB. In addition, TGF1 had no effect on IB and IB
levels. Electrophorectic mobility shift assays demonstrate that IL-1 is a potent inducer of NF-B translocation; however, TGF1 treatment did not result in shifting bands. Two adenovirus constructs were used to further clarify differences in TGF1- and IL-1-induced HAS1 activation. Overexpressing IB completely abolished the IL-1 effect on HAS1 but did not interfere with TGF1-induced HAS1 mRNA accumulation. Identical results were obtained when a dominant negative IKK was overexpressed. Interestingly, neither overexpression of IB nor of IKK had any effect on HAS2 and HAS3 mRNA levels. Taken together, HAS1 can be activated by distinct pathways; IL-1 utilizes NF-B, and TGF1 does not. Furthermore, HAS2 and HAS3 are activated without the involvement of NF-B. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Unfettered HA release, in association with inflammatory events, eventually results in degradation of the long chain HA molecules. Although little is known about the differences in physiological function of nondegraded HA molecules, it has been evident for some time that HA fragments exert a series of undesirable effects that might be linked to the progression of RA. Among these are the chemoattractant properties, induction of angiogenesis and the activation of proinflammatory genes (8, 9). For these reasons it seems vital to widen our understanding of the mechanisms involved in the regulation of the genes encoding HA synthases (HAS). Three genes, encoding for a plasma membrane protein, are responsible for HA synthesis (10). We demonstrated earlier that in type B synoviocytes (FLS) HAS2 and HAS3 are constitutively activated and that in this cell type HAS1 can be induced by a series of cytokines. We also showed that the p38 MAPK plays an essential role in cytokine induced HAS1 transcription (11).
Despite the considerable progress made in HA biology, still very little is known about intracellular signaling pathways participating in the regulation of these genes. Here we describe parts of our efforts to test the involvement of certain transcription factors in the activation of HAS genes. A broader insight into signaling pathways might provide hints at the function of the HAS genes as well as offer new ways to test the involvement of this important group of genes in RA and other diseases.
Many transcription factors have been said to take part in the regulation of genes. Among these factors, nuclear factor B(NF-B) seems to stand out for the central role this transcription factor plays in the activation of most proinflammatory genes (12, 13). With regard to RA, it has been recognized that NF-B also plays an essential role in both inflammatory and destructive mechanisms associated with joint destruction (14, 15). Taken together, a large body of research suggests that genes that depend on NF-B for their activation are linked to the onset and/or progression of inflammation. Based on this assumption, we investigated the requirement of NF-B activation for the genes encoding HAS in FLS. The experiments reported here are a continuation of our efforts to further understanding of the effects of TGF, IL-1, and TNF on HAS activation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(IL-1), TNF, and TGF1 were from Sigma. The antibodies for Western blots were from Cell Signaling (Cell Signaling, Bedford, MA). The antibodies for EMSA supershift experiments (c-Rel, RelB, p50, p52, and p65) were from Santa Cruz Biotechnology (Santa Cruz, CA). Oligonucleotides for CRE, NF-B, and AP-1 were from Promega (Mannheim, Germany). Oligonucleotides for SMAD1, SMAD3/4, STAT1, and STAT3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Culture—Human type B (fibroblast-like) synoviocytes were purchased from Dominion Pharmakine (Derio, Bizkaia, Spain). FLS were cultured as previously described (11). In brief, FLS were propagated in T75 tissue culture flasks or culture dishes (Iwaki, Funabashi, Chiba, Japan) (15 cm in diameter) in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), L-glutamine, and 50 units/ml penicillin/streptomycin. The medium was changed every 3 days. For experiments, FLS were detached using trypsin and transferred to 6- or 24-well plates (Iwaki). Viability of cells was confirmed by phase contrast microscopy and occasionally by staining cells with trypan blue.
Adenovirus-mediated Gene Transfer of Mutant mIKK and IB— Purified adenoviruses, containing the expression construct for dominant negative mutant IB kinase 2 (mIKK), IB, as well as green fluorescent protein (GFP), were a generous gift from Prof. R. de Martin (Medical University of Vienna, Vienna, Austria) (16–18). FLS were incubated with serial dilutions of each adenovirus to determine optimal concentrations for gene expression in this cell type. The transfection efficiency was monitored on a fluorescence microscope and/or by Western blot after 3 days in culture. In subsequent experiments a dilution of the stock material of 1:104 was used, and in some experiments a dilution of 1:105 was chosen.
Western Blot Experiments—For SDS-PAGE and Western blotting, the cells were washed twice in ice-cold PBS and subsequently dissolved in SDS sample buffer (62.5 mM Tris/HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% w/v bromphenol blue). For 3-cm culture dishes and 10-cm dishes, 100 and 500 µl of SB, respectively, was used. Aliquots of whole cell protein extract (10–25 µl/well) were separated on a mini gel (10%). The proteins were blotted onto polyvinylidene difluoride membranes (Amersham Biosciences) using a semi-dry apparatus (Bio-Rad). The blots were flashed with double distilled H2O, dipped into MetOH, and dried for 20 min before proceeding with the next steps. Subsequently, the blots were transferred to a blocking buffer solution (1x PBS, 0.1% Tween 20, 5% w/v nonfat dry milk) and incubated for 1 h. The membranes were then incubated with the appropriate diluted primary antibody in 5% bovine serum albumin, 1x PBS, and 0.1% Tween 20 at 4 °C overnight in a roller bottle. Following three washing steps in wash buffer (1x PBS, 0.1% Tween 20), the blots were incubated with appropriate secondary antibodies diluted in PBS. After 45 min of gentle agitation, the blots were washed five times for 5 min in wash buffer, and the proteins were made visible using either LumiGLO (New England Biolabs, Beverly, MA) or Renaissance Plus (PerkinElmer Life Sciences) and Kodak BioMax MR films.
RNA Isolation and Reverse Transcription—RNA isolation and reverse transcription were performed as described (11). Small aliquots of RNA were used to check the quality of RNA using agarose gel and ethidium bromide or Vistagreen (Molecular Probes, Eugene, OR) for visualization. First strand cDNA synthesis was performed exactly as described by the supplier of the RT-PCR Kit (Amersham Biosciences) using 1 µg of RNA/reaction.
RT-PCR and Data Analysis—Experiments on the block cycler were performed, analyzed, and quantitated as described (11). Real time RT-PCR and data analysis were done as follows: RNA was resuspended in water, quantitated on a spectrophotometer, and set to equal concentrations. Reverse transcription was performed using the Strata Script First Strand Synthesis System (Stratagene, Amsterdam, The Netherlands). Total RNA (500 ng) was transcribed, using random hexamer primers, and diluted with water to a final concentration of 250 ng to improve amplification efficiency. For real time RT-PCR, one-step SYBR Green RT-PCR amplification was conducted on a MX3000P real time PCR system (Stratagene), using Brilliant SYBR Green QPCR Master mix (Stratagene). After optimization of the primer pairs, RT-PCR was performed using the following standard conditions: initial denaturation for 10 min at 95 °C, denaturation for 10 s, annealing for 15 s at 57 °C, and extension for 15 s at 72 °C. Each RT-PCR experiment included a dissociation curve to verify the specificity of the amplicon, as well as no-template controls.
mRNA for hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT) was co-amplified and used as a control for quantification. Primers used in real time PCR experiments were selected based on published data (19, 20), ordered from MWG Biotech AG (Ebersberg, Germany), and dissolved at a concentration of 100 pmol/µl in Tris-EDTA. Primer sequences: HAS1 (sense primer), 5'-GAC TCC TGG GTC AGC TTC CTA AG-3'; HAS1 (antisense primer), 5'-AAA CTG CTG CAA GAG GTT AT TCC T-3'; HAS2: (sense primer), 5'-CTA TGC TTG ACC CAG CCT CAT C-3'; HAS2 (antisense primer), 5'-ACA CTG CTG AGG AAT GAG ATC CA-3'; HAS3 (sense), 5'-ACA GGT TTC TTC CCC TTC TTC C-3'; HAS3 (antisense), 5'-GCG ACA TGA AGA TCA TCT CTG C-3'; HPRT1: (sense), 5'-TGA CAC TGG CAA AAC AAT GCA-3'; and HPRT1 (antisense), 5'-GGT CCT TTT CAC CAG CAA GCT-3'. The length of amplified fragments are: HPRT, 93 bp; HAS1, 117 bp; HAS2, 107 bp; and HAS3, 166 bp. The correct length of the PCR product was confirmed by agarose gel electrophoreses. Standards and samples were assayed in a 25-µl reaction mixture containing 2x Brilliant SYBR Green QPCR Master Mix, 30 nM reference dye (ROX, carboxy-X-rhodamine), 1.5 µl of forward and reverse primer, cDNA (2.5 µl), and double distilled H2O.
Electrophoretic Mobility Shift Assay—Nuclear extracts from FLS were prepared as described (21). The double-stranded oligonucleotides used in all of the experiments were end-labeled using T4 polynucleotide kinase and [
-32P]ATP. After labeling and purification by chromatography, 5 µg of nuclear extract was incubated with 100,000 cpm of labeled probe in the presence of 1.5 µg of poly(dI-dC) at room temperature for 20–30 min followed by separation of this mixture on a 6% polyacrylamide gel in Tris/glycine/EDTA buffer at pH 8.5. As also published previously (21), a series of control experiments were performed that confirmed the specificity of EMSA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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IB Degradation Following IL-1, TNF, and TGF Treatment—In resting cells, IB is bound to the NF-B protein complex and plays an important role in preventing its translocation to the nucleus. Activation of the NF-B pathway is essential for the activation of most proinflammatory genes (22). We used Western blot experiments to investigate whether exposure of FLS to IL-1 (1 ng/ml), TNF (5 ng/ml), and TGF (1 ng/ml) will lead to the degradation of IB. Presented in the upper panel of Fig. 2A are the results of a representative experiment where cells were left untreated (lane MED) or stimulated with IL-1 (lane IL-1) or TGF (lane TGF) for 10 min. As shown, treating cells with IL-1 results in complete degradation of IB, whereas TGF treatment has no apparent effect on IB.
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B Phosphorylation—IB degradation is preceded by phosphorylation of IB, tagging it for subsequent degradation by specific enzymes. Measuring phosphorylation of IB provides another means to assess the involvement of the NF-B signaling pathway by a given stimulus. FLS were treated with IL-1 and TGF for 10 min. Whole cell protein extracts were separated by PAGE. The results of such Western blots, using phospho-specific anti-IB antibodies, reveal that stimulation with TGF does not result in phosphorylation of IB. As demonstrated in the lower panel of Fig. 2A, treating FLS for 10 min with IL-1 leads to a strong phosphorylation specific signal. Contrary to the result obtained with IL-1, IB phosphorylation was detected neither in TGF-treated cells nor in resting cells.
It is conceivable that there are differences in the time course of IB degradation induced by differences in the upstream signaling pathways utilized by these two stimuli. Shown in Fig. 2B is a representative experiment where IB degradation was monitored for an extended period of time. Similar to IL-1, TNF treatment of FLS for 10 min also results in complete degradation of IB within a very short time frame. More importantly, TGF treatment did not result in reduced IB levels when those levels were monitored for up to 1 h. Shown in this particular experiment are the IB levels at 0, 10, 20, and 50 min after TGF (1 ng/ml) stimulation. Equal loading is demonstrated by staining a second blot with a control antibody (labeled Contr).
Other members of the IB protein family might be involved in TGF-induced signaling. A Western blot experiment investigating the effect of TGF treatment on IB,IB, and IB is shown in Fig. 2C. FLS were exposed to TGF and TNF, respectively, for 10 min. Similar to the results of the Western blot experiment shown in Fig. 2B, TNF treatment for 10 min resulted in complete degradation of IB but left IB and IB unaffected. TGF treatment, on the other hand, had no effect on any of the IB proteins monitored.
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B, and TGF Treatment Is Without Effect—A number of studies have reported that NF-B translocation into the nucleus could be detected without observing prior IB degradation, leaving the possibility that TGF could activate the NF-B pathway in a similar way. To monitor the translocation of the transcription factor NF-B into the nucleus, EMSA experiments were performed. Shown in Fig. 3A is one of two EMSA experiments where FLS were stimulated for 0, 15, 30, 45, 60, 75, and 90 min, respectively. Indicated on the left side are the stimuli used. The label FP indicates the lane of the free probe. The specific bands for CRE and NF-B as well as the position of a non-NF-B band (marked as NS) are indicated on the right side of the blot. As shown in Fig. 3A, IL-1 stimulation not only results in IB degradation but also in a swift translocation of this transcription factor into the nucleus. Maximal levels of NF-B-DNA interaction can be seen at 30–40 min after stimulation, but even 90 min later, DNA-NF-B complexes can be observed that are still considerably higher than in untreated cells. As shown in the lowest section of Fig. 3A, specific NF-B-DNA complexes are low in unstimulated cells, and no changes in NF-B-DNA complex formation can be seen in nuclear extract isolated from TGF-treated cells. To monitor equal nuclear protein concentration in EMSA, CRE-specific EMSA were routinely performed and are shown in the top panel, labeled CRE.
Shown in Fig. 3B are experiments set up to analyze the composition of the shifted NF-B-DNA complex and to confirm the specificity of the EMSA experiments. Only low basal protein-DNA interactions are seen in unstimulated FLS (lanes MED). Besides using extract of unstimulated cells as controls, FLS were treated with IL-1 (5 ng/ml) for 45 min, after which nuclear protein extract was isolated und used for a series of experiments. First, supershift experiments were performed utilizing antibodies specific for NF-B family members. Aliquots of nuclear extracts of IL-1-treated FLS were used without the addition of antibodies (Fig. 3B, none) or were preincubated with antibodies directed against NF-B p50, p65, p52, c-Rel, and RelB for 30 min at room temperature prior to the addition of labeled oligonucleotides. Such experiments reveal the presence of the prototypical NF-B p65/p50 heterodimer complex in IL-1-stimulated FLS. Also shown in Fig. 3B are experiments demonstrating that the addition of 500-fold excess of unlabeled homologous NF-B oligonucleotides are able to compete for NF-B-DNA binding (lane cold NF-B). On the other hand, the addition of 500-fold excess of unrelated AP-1-oligonucleotides (lane cold AP-1) was without any effect on NF-B-DNA interactions. Similarly, adding similar amounts of unlabeled SP1 or CRE oligonucleotides was without effect on NF-B-DNA interactions (data not shown).
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The activation of members of the SMAD family is among the best studied pathways involved in TGF-mediated intracellular signaling events. Innumerable studies describe the utilization of SMAD proteins as the main intracellular signaling pathways utilized by this compound (26–28). For that reason, SMAD3 is sometimes also referred to as TGF response effector SMAD3. In an attempt to investigate whether activation of SMAD proteins might account for the effect of TGF on HAS1 in FLS, the activation and nuclear translocation of SMAD1, SMAD3, and also that of STAT1 and STAT3 proteins were evaluated by EMSA. Shown in Fig. 3C is one of three EMSA experiments where FLS were left untreated or were exposed to TGF (1 and 10 ng/ml, respectively) for 45 min. Nuclear protein extract and EMSA were performed as described under "Experimental Procedures." A weak protein-DNA complex can be observed in unstimulated cells (Fig. 3, B and C, MED). Exposing FLS to 1 ng/ml TGF for 45 min did not result in a significant increase in SMAD3/4-DNA complexes. Interestingly, increasing the concentration of TGF to 10 ng/ml resulted in the activation of SMAD3/4. Similarly, STAT3 activation was not detected in FLS stimulated with 1 ng/ml TGF, yet again, using 10-fold higher concentrations of TGF led to a readily detectable increase in STAT3-DNA complexes. Although SMAD3/4 as well as STAT3 were activated in FLS stimulated with 10 ng/ml of TGF, no activation of SMAD1 and STAT1 could be observed in similar EMSA experiments using oligonucleotides resembling consensus SMAD1 and STAT1 sequences; neither 1 nor 10 ng/ml TGF treatment resulted in significant activation of these transcription factors (data not shown). Also included in Fig. 3C (far right panel) is yet another control experiment, demonstrating that PMA results in the activation of NF-B when used at a concentration (2.5 ng/ml) that significantly increases HAS1 mRNA accumulation in FLS (11).
Overexpressing IB and Dominant Negative mIKK Prevents IL-1-induced HAS1 Activation but Does Not Effect TGF-induced HAS mRNA Accumulation—As demonstrated many times, nuclear translocation of NF-B can be prevented by overexpression of a mutant version of IKK (16, 29). Such a measure seems well suited to clarifying the involvement of NF-B in IL-1- and TGF-induced HAS activation. FLS were transfected with adenovirus constructs containing either a mutant IKK or a GFP expression sequence as described under "Experimental Procedures." Shown in Fig. 4A is such a representative experiment performed in duplicate. As indicated for Fig. 4A, FLS (nontransfected or IB transfected) were left untreated (MED) or stimulated with IL-1 and TGF, respectively, for 10 h. The effect of preventing NF-B translocation was studied by comparing induced and noninduced HAS1, HAS2, and HAS3 mRNA levels.
Such experiments demonstrated that overexpression of IB affected only HAS1 mRNA levels in significant ways. Stimulating FLS with IL-1 and TGF treatment results in >50- and >30-fold higher levels of HAS1 mRNA, respectively. More importantly, as shown in the panel labeled HAS1, whereas the IL-1 effect on HAS1 mRNA levels in cells expressing IB is completely abolished, no inhibitory effect could be observed on TGF-induced HAS mRNA accumulation. As shown in Fig. 1, HAS2 and HAS3 mRNA levels in resting FLS are relatively high. Interestingly, overexpression of IB had neither an effect on the high basal levels of these genes, nor were mRNA levels of HAS2 and HAS3 significantly altered in FLS that were stimulated with IL or TGF (Fig. 4A, panels HAS2 and HAS3, respectively).
Activation of IKKs upstream of IB plays an important role in the signaling cascade leading to the degradation of IB (30, 31). We used a mutant IKK2 adenovirus expression construct to validate data obtained using the IB construct and to further dissect possible differences in signaling pathways in IL-1-induced versus TGF-induced HAS activation. Parts of 6-well tissue culture dishes containing FLS grown to high density were treated with the mIKK adenovirus for 3 days. Subsequently, FLS were left untreated (lane MED) or were stimulated with IL-1 and TGF, respectively, for 10 h, at which time the experiment was terminated and RNA was extracted. A comparison of the data presented in Fig. 4B reveals similar effects as observed using the IB construct to block NF-B-mediated signaling. As demonstrated here again, treatment of FLS with IL-1 and TGF results in >50- and >30-fold higher levels of HAS1 mRNA, respectively, whereas levels of HAS2 and HAS3 mRNA are not significantly affected. Similar to the results presented in Fig. 4A using an IB construct, overexpression of mIKK also leads to a complete inhibition of IL-1-induced HAS1 activation. Furthermore, like high levels of IB, overexpression of mIKK has no effect on TGF-induced activation of this gene. Again, HAS2 as well as HAS3 mRNA levels are not significantly affected by the presence of high amounts of mIKK in the cytoplasm.
Real Time PCR, Standard Curves, and PCR Analysis—Pictures depicting representative real time PCR experiments for HAS1, HAS2, and HAS3 as well as HPRT are given in Fig. 5. The name of the gene illustrated is given on the top of each panel. The lower panels show amplification curves, and the upper panels show the standard curves used for quantification (calculation of x-fold induction).
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B-mediated inhibition of IL-1-induced HAS1 activation. For 3 days, FLS were incubated with adenovirus constructs (GFP, mIKK, and IB, respectively) after which cells were exposed to IL-1, to TGF, or were left untreated (MED). mRNA levels were quantitated by real time PCR. As demonstrated in the section labeled IL-1, overexpression of mIKK as well as IB completely abolished the 60-fold induction. More importantly, overexpression of GFP in these cells had no effect on HAS1 mRNA levels induced by IL-1 treatment. Fig. 6A demonstrates again that TGF-induced HAS1 mRNA levels are not affected by overexpressing mIKK or IB, respectively. This figure clearly demonstrates that HAS1 mRNA levels are affected neither by the presence of the virus construct itself, nor by the high concentrations of GFP present in transfected cells.
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B protein in the cytoplasm of cells transfected with IB adenovirus expression construct. In this particular experiment, FLS (two wells each) were left untreated (lanes MED), transfected with the IB adenovirus construct (lanes V-IB), or with the GFP virus construct (lanes V-GFP) and incubated for 3 days. Whole cell extract was prepared and separated by SDS-PAGE. As demonstrated in this figure, levels of IB in cells exposed to the IB adenovirus construct are significantly higher than those in untreated cells. Furthermore, treating cells with the GFP adenovirus construct does not lead to changes in the IB levels. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B in induced and noninduced HAS activation in in vitro experiments using FLS.
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, IL-1, IL-8, TGF, and TNF (11). HAS1 mRNA levels are very low in resting FLS but mRNA for HAS2 and HAS3 is readily detectable in resting cells. Interestingly, HAS1 was the only gene whose mRNA levels were significantly altered in response to the above stimuli. Here we report that the gene HAS1 can be activated in a NF-B-dependent as well as in a NF-B-independent manner. Although the utilization of the NF-B pathway is a prerequisite for IL-1-induced HAS1 activation, TGF-induced activation of HAS1 occurs independently of NF-B. These conclusions are based on a series of experiments analyzing the activation of the NF-B pathway in response to TGF and IL-1.
As shown here, stimulating FLS with IL-1 resulted in rapid phosphorylation and subsequent degradation of IB. In contrast, TGF treatment had no effect on IB. Furthermore, TGF treatment did not lead to the phosphorylation of IB, which in most cases is a prerequisite for subsequent IB degradation. Because it is feasible that in FLS other IB members might play a dominant role in the retention/release of NF-B, the effect of TGF on IB and IB was investigated as well. Similar to IB levels, TGF treatment of FLS did not lead to any significant changes in cytoplasm levels of those IB proteins.
Although in most reports IB degradation has been shown to be essential for the release and the subsequent translocation of the NF-B complex into the nucleus, there are a few reports demonstrating the activation of NF-B without prior degradation of IB (35). To test the possibility of TGF-induced NF-B activation without prior degradation of IB, EMSA experiments were performed. The results of these experiments clearly demonstrate that although IL-1 induces the translocation of NF-B into the nucleus as well as subsequent binding to oligonucleotides resembling consensus NF-B binding sites, TGF does not.
EMSA are to a certain degree artificial systems that might not entirely mimic in vivo conditions. We therefore resorted to using two adenovirus constructs that would allow us to validate the interpretation of Western blot and EMSA experiments. First, FLS were transfected with an adenovirus construct containing the expression sequence for IB. As demonstrated in Fig. 6B, at the time of the experiment, high levels of IB could be demonstrated by Western blot. More importantly, overexpressing IB led to complete inhibition of IL-1-induced HAS1 transcription. On the other hand, the 
30-fold higher HAS1 mRNA levels induced by TGF treatment were not reduced. Identical results were obtained when instead of the IB construct, an adenovirus gene transfer system was used that results in the expression of a mutant kinase-negative version of IKK. That this kinase, like IB, plays an essential role in the NF-B-mediated signaling pathway has been shown before (30, 31).
The outcome of these experiments yet again demonstrates the involvement of NF-B in IL-1-induced HAS1 transcription and also bolsters the conclusion that TGF-induced signaling leading to HAS1 activation indeed occurs independently of NF-B. The effects of overexpression of IB and mIKK are attributable only to the elevated levels of these proteins and are not mediated either by nonspecific effects of the virus construct itself or by the transfection conditions used. This conclusion is supported by the demonstration that the adenovirus-GFB construct affects neither IL-1- nor TGF-induced HAS1 activation.
We described earlier that the maximal level of HAS1 mRNA can be observed in FLS stimulated with TGF at a concentration of 0.5–1 ng/ml (11). We also demonstrated that increasing the amount of TGF to 10 ng/ml not only reduced the stimulatory effect on HAS1 significantly but also reduced levels of the constitutively elevated HAS3 mRNA in FLS. That the TGF effect greatly depends on the concentration of TGF as well as on the effector cells used has been shown in numerous studies (36–38). It is also of interest that Westergren-Thorsson et al. (39) reported that TGF enhances HA production in lung but not in skin fibroblasts. Although Sugiyama et al. (40) reported up-regulation of the gene HAS1 in skin fibroblasts stimulated by TGF, several reports confirm our findings and reported TGF-induced activation of HA release in FLS (41, 42). Others also studying the effect of TGF on HA reported no effect of TGF on HA or even demonstrated TGF-induced inhibition of HA release (43). Although likely, it is currently not clear whether the above discrepancies can be explained solely by differences in the concentration, the length of exposure, or the biological activity of TGF used.
Because TGF does not activate the NF-B pathway, we performed a series of experiments aimed at a better understanding of the mechanisms that account for TGF induced HAS1 activation. Although PKC has been implicated in TGF-induced signaling pathways, our experiments blocking PKC with staurosporine indicate that in FLS, PKC is not involved in TGF-mediated HAS1 activation. Similarly, a cAMP-dependent protein kinase as well as a protein kinase G inhibitor also failed to reduce TGF-mediated HAS1 mRNA accumulation. In addition, as demonstrated by the representative EMSA experiment shown in Fig. 3C, SMAD3/4 as well as STAT3 can be activated in FLS. However, at the concentrations of TGF that lead to induction of HAS1, TGF neither activated SMAD3/4 nor STAT3. This is surprising, because especially proteins of the SMAD family have been described to be closely associated with TGF-induced intracellular signaling (26, 28, 44).
The fact that in FLS, HAS2 and HAS3 are constitutively activated speaks against a role of NF-B in the activation of these genes. With few exceptions e.g. in B-cells, NF-B is not activated in resting cells (45). The data presented here also support the conclusion that in FLS neither HAS2 nor HAS3 depend on the activation of NF-B. As demonstrated in Fig. 1, mRNA levels of HAS2 and HAS3 in unstimulated FLS are indeed high, but EMSA experiments do not demonstrate significant NF-B-DNA interactions in nuclear proteins isolated from resting FLS. Furthermore, neither HAS2 nor HAS3 mRNA levels decrease in response to obstruction of the NF-B signaling pathway through overexpression of mIKK and IB, respectively.
The very nature of the HA molecules has complicated and considerably hampered investigations in functional differences of the HAS gene products. Therefore, very few studies directly address biological effects that might be attributable to dissimilarities of HA molecules. Nevertheless, we have previously speculated about the fact that HAS1 is the sole HA encoding gene that readily responds to a series of proinflammatory cytokines (11). If a generalization from published data is legitimate, and the assumption of the NF-B dependence of proinflammatory genes also holds true for HA encoding genes, HAS1 would fulfill yet another criterion to justify its classification as a proinflammatory gene. Currently it is unclear which of the intracellular signaling pathways are utilized in NF-B-independent HAS1 activation. Nevertheless, similar phenomena have been observed by others studying mediators of inflammation. It has been shown, for example, that TNF, IL-1, IL-6, and IL-8 can also be regulated in both NF-B-dependent and NF-B-independent ways (46).
The critical role of IL-1 in the propagation of RA and inflammation in general is well accepted (47, 48). On the other hand, participation of TGF in inflammatory processes is less recognized, although certain observations clearly link TGF to inflammation (49, 50). With regard to RA, not only are there reports demonstrating that TGF is indeed abundant in the rheumatoid synovium (51), it has also been shown that TGF is able to exert a series of detrimental effects on FLS isolated from RA patients. Among these is the activation of IL-1, TNF, IL-8, MIP1, and MMP-1. In addition, most of these effects of TGF seem specific to the arthritic synovial fibroblasts (52).
Concentrations of HA are considerably higher in the affected joints and plasma of patients suffering form RA than in both healthy subjects and in osteoarthritis patients (7). Still, to date, no causal link between up-regulation of HAS genes and the subsequent infiltration of leukocytes in affected joints in RA has been established. It nevertheless seems plausible that unfettered HA release contributes to initiation and/or propagation of RA. Such a view is also strongly supported by the recent demonstration that HA can cause all classical signs of RA (53). Taken together, these data seem to support our working hypothesis that unregulated HA metabolism does play a significant role in initiation and/or progression of RA.
In summary, we demonstrate the existence of two independent pathways of HAS1 activation. In addition, our data also seem to rule out a role of the transcription factor NF-B in the activation of the genes HAS2 and HAS3. The presented data might offer new insight in TGF-induced signaling pathways and contribute to a better understanding of regulatory mechanisms in HA biology.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Ludwig Boltzmann Institute for Rheumatology and Balneology, Kurbadstrasse 10, 1100 Vienna, Austria. Tel.: 43-1-68-00-99-231; Fax: 43-1-68-00-99-234; E-mail: karlms{at}excite.com.
2 The abbreviations used are: HA, hyaluronan; HAS, hyaluronan synthase; EMSA, electrophoretic mobility shift assay(s); NF-B, nuclear factor B; IB, inhibitor B; IKK, IB kinase; mIKK, mutant IKK; FLS, fibroblast-like synoviocytes (type B synoviocytes); TGF, transforming growth factor; IL, interleukin; RA, rheumatoid arthritis; MAPK, mitogen-activated protein kinase; TNF, tumor necrosis factor; GFP, green fluorescent protein; PBS, phosphate-buffered saline; RT, reverse transcription; HPRT, hypoxanthine-guanine phosphoribosyl transferase; CRE, cyclic responsive element; PMA, phorbol myristate acetate; PKC, protein kinase C; STAT, signal transducers and activators of transcription; SMAD, Sma- and Mad-related proteins.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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)) are indicated on the right side of each panel. Indicated on the left are the oligonucleotides used (NF-
