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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2001 as doi:10.1096/fj.00-0543fje. |
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Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA
2Correspondence: Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. E-mail: jane.ehrke{at}roswellpark.org
SPECIFIC AIMS
TIP-B1, a 27 kDa protein, was first identified by our laboratory as a cytosolic protein produced by cells pretreated with low, nontoxic levels of tumor necrosis factor (TNF) that protects cells from the cytotoxicity induced by high, toxic levels of TNF. Two primary aims of these studies were to establish whether or not untreated normal human dermal fibroblasts (NHDF) express TIP-B1 and, if so, to determine its cellular localization. A third aim was to determine whether there were changes in TIP-B1 levels or location during protective TNF pretreatment.
PRINCIPAL FINDINGS
1. Anti-TIP-B1 antisera detect TIP-B1 in membrane and cytosolic
protein preparations from NHDF
We used two antisera in these studies: one raised against purified
native TIP-B1 (anti-TIP-B1) and an antiserum raised against a 14 kDa
recombinant protein (rTIP-B1partial) that is the carboxyl half of
TIP-B1 (anti-rTIP-B1p). Both antisera recognized a 26/27 kDa protein
doublet in Western blots of NHDF cytosolic proteins. Excess rTIP-B1p
blocked binding of anti-rTIP-B1p to these 26/27 kDa proteins in Western
blots. Preimmune sera did not react with 26/27 kDa proteins and there
was little reactivity of anti-TIP-B1p antiserum with proteins other
than those with an Mr of 26/27 kDa
when NHDF whole-cell lysates were examined. These findings verified the
specificity of the reactivity of the antibodies with the TIP-B1 protein
and established that it is constitutively expressed in NHDF. Using
anti-rTIP-B1p antiserum, TIP-B1 was detected in Western blots of NHDF
cytosol and membrane subfractions, isolated by either differential
centrifugation or digitonin-triton sequential detergent extractions. An
immunoreactive 27 kDa protein was not detected, however, in the
cytoskeletal fraction (both digitonin and triton insoluble). The 26/27
kDa protein doublet was reduced to a single immunoreactive 25 kDa
protein band after treatment with peptide-N-glycanase F, suggesting
that the 26 and 27 kDa proteins are different glycosylated forms of
TIP-B1. Immunocytochemistry of formaldehyde-fixed, paraffin-embedded
NHDF cell pellets confirmed reactivity of anti-rTIP-B1p antiserum with
plasma membrane and cytoplasmic elements.
2. Pretreatment of NHDF with low concentrations of TNF is
protective against the cytotoxicity of the combination of TNF and
cycloheximide
Consistent with the constitutive expression of TIP-B1, NHDF are
resistant to TNF as a single agent. However, NHDF are sensitive to the
combination of TNF (200 ng/ml) with cycloheximide (100µg/ml), a
protein synthesis inhibitor. Pretreatment of NHDF with low
concentrations (i.e., 0.4–40 ng/ml) of TNF protected 75–90% of the
NHDF from TNF-mediated lysis of the combination. Protection of the
cells was detected after 2 h of pretreatment, and protection
reached maximum levels by 4 h of pretreatment.
3. Changes in cytosolic and membrane TIP-B1 protein levels precede
a rise in cytosolic protective activity during TNF pretreatment
The levels of TIP-B1 expression in NHDF were determined at various
times during induction of protection by low concentrations of TNF.
Western blot analyses of cytosolic preparations from NHDF cells after
TNF pretreatment (20 ng/ml) revealed that detectable cytosolic TIP-B1
levels start increasing at 15 min, are near maximal (1.8-fold) at
2 h of pretreatment, and gradually decrease thereafter (Fig. 1
). Within the first 30 min of pretreatment, the levels of detectable
TIP-B1 in membrane fractions decreased; subsequently, levels greater
than those at time 0 were detected. The percentage of cells exhibiting
strong cytoplasmic staining, as assessed by immunocytochemistry,
increased from 4.5 ± 2.6% to 95.8 ± 3.3% during the first
30 min, decreasing after that point. Western blot analyses of
whole-cell lysates also showed the increase and then decrease in
cellular TIP-B1 levels during TNF pretreatment, closely paralleling
changes in cytosolic TIP-B1 (Fig. 2A
).
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The original identification of TIP-B1 was facilitated by the fact
that TNF-sensitive cells cultured with cytosol from cells rendered
resistant to TNF were protected from TNF-induced lysis. The cytosolic
preparations from NHDF examined by Western blot analysis (Fig. 2A
) were also evaluated for the ability to protect NHDF from
TNF (Fig. 2B
, solid circles). The sustained increase in
protective activity in the cell cytosol was preceded by the increase in
TIP-B1 levels (Fig. 2)
. This activity began rising between 1 and 2 h of TNF pretreatment, reached a plateau by 6 h, and persisted
until 18 h. The kinetics of this increase in cytosolic protective
activity paralleled the increase in cellular protection (Fig. 2B
, solid triangles) induced by TNF pretreatment (Fig. 2)
.
4. TIP-B1 mRNA does not increase substantially during the first
hour of pretreatment
TIP-B1 mRNA in NHDF, as assessed by Northern blot and
normalization to GAPDH mRNA, did not increase significantly during the
first hour of TNF pretreatment. TIP-B1 mRNA levels did increase
slightly at later times, maximizing (1.6-fold) at 3 h of TNF
pretreatment.
CONCLUSIONS AND SIGNIFICANCE
This study is significant because it is the first report of the
cellular localization of TIP-B1. These data demonstrate that TIP-B1 is
present not only in the cytosol, but also in the plasma membrane of
NHDF cells. It can be proposed that these are the sites where the
protein is active. In addition, the activity of TIP-B1 may be regulated
by TIP-B1 protein levels at these sites. Pretreatment with low nontoxic
concentrations of TNF induces resistance in NHDF to TNF-mediated
cytotoxicity. TIP-B1 protein levels begin changing in response to TNF
within 15 min of initiation of treatment; they increase in the cytosol
and first decrease and then increase in the membrane (Fig. 1)
. The
mechanism by which NHDF regulates TIP-B1 levels in response to TNF is
unknown. Although Northern blot analysis indicated TNF pretreatment
increased TIP-B1 mRNA to the same degree as it increased TIP-B1
protein, this did not occur until the cells had been treated with TNF
for 3 h. It is suggested, therefore, that one or more
post-transcriptional mechanism are involved.
The changes in TIP-B1 levels are followed by an increase in protective
activity in the cytosol; however, TIP-B1 protein levels begin to
decrease while protective activity in the cytosol is still rising (Fig. 2)
. These observations suggest that changes in TIP-B1 levels may be a
trigger for subsequent downstream protective events in the cytoplasm
(Fig. 3
). These events may include the production or activation of secondary
protective factors in the cytoplasm, although the exact nature of these
factors has not yet been elucidated.
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TNF is known to induce the production of TNF-protective proteins such as manganese superoxide dismutase (MnSOD), plasminogen activator inhibitor-type 2 (PAI-2), and A20 protein. There is evidence that, unlike TIP-B1, these proteins are induced at the mRNA level. There are also functional distinctions between these proteins and TIP-B1. Thus, there is no evidence in the literature that MnSOD, PAI-2, or the A20 protein protect when added exogenously to cells; TIP-B1, however, does. Furthermore, MnSOD is increased in mitochondria and not in the cytoplasm. Like TNF, pretreatment with interleukin 1 (IL-1) can protect NHDF from the combination of TNF and cycloheximide (data not shown), and IL-1 has been shown to be produced by fibroblasts after TNF treatment. However, this IL-1 was membrane associated and not cytosolic. Therefore, it can be proposed that an unidentified factor or combination of factors is the actuator of TNF resistance that is downstream of changes in TIP-B1 levels. The decrease in TIP-B1 levels after 2 h of pretreatment could be explained by degradation of TIP-B1 or the modification of TIP-B1 into an undetectable form; there is no evidence yet to discriminate between these two mechanisms.
In conclusion, the studies presented here clearly establish that TIP-B1 is located in the cytosol and plasma membrane of NHDF. They demonstrate that TIP-B1 protein levels increase preceding a sustained increase in TNF-protective activity in the cytosol, which suggests that a secondary protective factor may be involved. The studies also indicate that the increase in TIP-B1 is most likely mediated at a post-transcriptional point.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0543fje ; to cite this article, use FASEB J. (March 5, 2001) 10.1096/fj.00-0543fje 
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–) are
superimposed (B) with the levels of protection afforded
NHDF by preincubation with the same cytosolic preparations at 15
µg/ml (–•–) and with the level to which the NHDF preincubated
with the 20 ng/ml TNF were protected (-
-). To assess the protective
effect, NHDF at 104 cells/well were pretreated with
cytosolic proteins or TNF for 18 h, then incubated in 200 ng/ml
TNF + 100 µg/ml CHX for 18 h. MTT assays were used to estimate
cell survival at the end of the treatment period. These results are
representative of 3 independent experiments.