The activities of MAPKs are necessary for the initiation of the innate immune
response. Both the activation of transcription factors by phosphorylation and the
stabilization of ARE-containing mRNA can help cells to produce pro-inflammatory
mediators or cytokines. The activations of MAPKs are turned off within several minutes,
and then the mRNAs of those pro-inflammatory mediators or cytokines will be
degraded rapidly. Some negative regulators such as Mkp-1 (phosphatase) and Ttp
(mRNA decay mediator) can help cells to return to the resting condition. Hence, the
gene expressions should be tightly controlled in both transcription and
post-transcription levels in the innate immune response.
We are the first to observe the protein expression profiles of endogenous TTP
family proteins during the period of LPS-stimulation in mouse macrophage RAW264.7
cells. There are two interesting observations. One is the protein expression profiles of
these three TTP family members were different, and the other is the uncorrelated mRNA
and protein expression profiles of Zfp36l1 and Zfp36l2.
Ttp was induced after LPS-stimulation, and Zfp36l1 and Zfp36l2 were constitutive
expressions (Figure 1A). This observation indicates that Zfp36l1 and Zfp36l2 may play
important roles in the resting condition, and the low expressions of pro-inflammatory
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mediators or cytokines are maintained by the mRNA destabilized abilities of Zfp36l1
and Zfp36l2.
As shown in Figure 1B, the mRNA expressions of Zfp36l1 and Zfp36l2 were
down-regulated after LPS-stimulation, but the protein expressions of Zfp36l1 and
Zfp36l2 were consistent. The mRNA expression profiles of Zfp36l1 and Zfp36l2 were
uncorrelated with their protein profiles after LPS-stimulation (Figure 1B). Some similar
observations of mRNA expression profiles of Zfp36l1 and Zfp36l2 were also reported
by Liang et al. [48] and Cao et al. [49]. This uncorrelated relationship implys the
expressions of Zfp36l1 and Zfp36l2 are regulated in the translational and
post-translational levels. The polysome analysis of their mRNA during LPS-stimulation
can be performed to verify the translational regulation. Furthermore, the
post-translational modifications such as phosphorylation (Figure 1C) may alter protein
stability [25]. Zfp36l1 and Zfp36l2 may be more stable in the hyper-phosphorylated
forms than the hypo-phosphorylated forms. Thus, their protein expression levels are
near consistent, even their mRNAs are decreased after LPS-stimulation.
To identify the possible mRNA targets of Zfp36l1 and Zfp36l2 in resting
macrophages, we knocked down Zfp36l1 and Zfp36l2 by using Lentivirus-carrying
shRNA. Based on previous reports, the candidates of mRNA targets are chosen by the
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ARE number in their 3’UTR, the associations with Ttp, and their mRNA expression
profiles which were categorized to the immediate-early genes [39,50].
In our study, the RNA and protein levels of Mkp-1 were increased in Zfp36l1,
Zfp36l2, and dual knockdown cells (Figure 2A, 2B). In addition, the RNA and protein
levels of Cox-2 were increased in the cells which Zfp36l2 was knocked down (Figure
2A, 2D). The mRNA expression levels of Mkp-1 and Cox-2 were increased through the
mRNA stabilities when Zfp36l1 and Zfp36l2 were knocked down, which were
correlated with the typical function of Zfp36l1 and Zfp36l2 in mRNA destabilization
(Figure 2C, 2E).
Tnf-α is one of the well-known targets of TTP family proteins. Much to our
surprise, the mRNA expressions of Tnf-α were decreased in all knockdown cells (Figure
2F). This result may be due to the importance of the transcription regulation of Tnf-α
mRNA controlled by activation of p38 MAPK [35]. Therefore, the increase of Mkp-1
expression in Zfp36l1 and Zfp36l2 knockdown cells repressed the activity of p38
MAPK (Figure 6A), which down-regulated the Tnf-D mRNA expression. Similarly, the
mRNA expression of Ccl-2 is also activated by p38 MAPK [51]. However, the mRNA
expression of Ttp and Icam-1 were no significant differences whether Zfp36l1 and
Zfp36l2 were knockdown or not (Figure 2F). The possible explanation is that the
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expressions of their mRNAs are controlled equally in both transcriptional and
post-transcriptional levels.
The mRNA targets of TTP family proteins are not all the same [52], although their
RNA binding domains are highly conserved. In Figure 2A and 2D, Cox-2 was the
Zfp36l2-specific mRNA target. However, the activity of luciferase containing Cox-2
3’UTR was down-regulated by both over-expressed Zfp36l1 and Zfp36l2 (Figure 3B).
This result indicates that both of Zfp36l1 and Zfp36l2 can recognize and destabilize the
Cox-2 mRNA in HEK 293T cells. However, overexpression of Zfp36l1 and Zfp36l2 in
HEK 293T cells may not reflect their “real” functions and the “real” conditions in
RAW264.7 cells. The RNA pull-down assay was shown that both of Zfp36l1 and
Zfp36l2 could associate with Cox-2 3’UTR in RAW264.7 cells (Figure 3C). Both of
Zfp36l1 and Zfp36l2 do not have enzyme activity of mRNA decay, so the difference of
their associated proteins may alter their functions. Mass spectrometry may be used to
identify the associated proteins of Zfp36l1 and Zfp36l2, and the detailed regulation can
be further studied. In conclusions, the mRNA expression of Cox-2 is Zfp36l2-specific
regulation in the post-transcriptional level.
The rapid mRNA activation of Mkp-1 after the LPS-stimulation was regulated
through mRNA stability (Figure 4A, 4B). In Figure 4C, the interaction between Zfp36l1
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and Mkp-1 3’UTR was consistent during LPS-stimulation, but the interaction between
Zfp36l2 and Mkp-1 3’UTR was variable with the protein expression of Zfp36l2. This
result suggests that the decline of Zfp36l2 on Mkp-1 3’UTR stabilizes Mkp-1 mRNA.
Knockdown of Zfp36l1 and Zfp36l2 increased the mRNA expression of Mkp-1 through
RNA stability in the early stage of LPS-stimulation (Figure 5A, 5B). This result
confirms that the mRNA activation of Mkp-1 is regulated by Zfp36l1 and Zfp36l2 after
LPS-stimulation.
In addition, the activation of Mkp-1 is also regulated by MAPKs in both
transcriptional and post-translational levels. The activation of ERK signaling pathway
increases the transcription of Mkp-1 [53], and the activation of ERK and p38 MAPK
signal pathway can increase the protein stability of Mkp-1 by phosphorylation [54,55].
Associated proteins may change the functions of Zfp36l1 and Zfp36l2. We were
the first to observe that TTP family proteins, Zfp36l1 and Zfp36l2, could associate with
deadenylase Caf1a and degrade mRNA targets by removing poly-A tails (Figure 4C).
However, Zfp36l1 could be phosphorylated after LPS-stimulation and form the complex
with 14-3-3 (Figure 4D). This complex may repress the function of Zfp36l1 [26], and
Mkp-1 mRNA can be stabilized.
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Knockdown of Zfp36l1 and Zfp36l2 increased the protein expression of Mkp-1
(Figure 6B), and the activity of p38 MAPK was down-regulated in the resting condition
(Figure 6A). Therefore, p-38 mediated mRNA expressions of Ttp and Tnf-α were
repressed (Figure 6C). According to the results, we proposed the following model for
the mechanism (Figure 7). In the resting condition, Zfp36l1 and Zfp36l2 destabilize
mRNA of Mkp-1, and the cells are sensitive to the stimuli such as LPS under the low
expression of Mkp-1. In LPS-stimulation, the induction of Mkp-1 mRNA is done by
hyper-phosphorylated Zfp36l1 which losing its ability of mRNA decay and decreasing
the expression of Zfp36l2.
A number of important ARE-containing genes are commonly involved in
inflammation and cancer [45]. Both MKP-1 and COX-2 have been reported playing
important roles in human cancers. MKP-1 is over-expressed in many human cancer cell
lines, including breast, lung, prostate, ovarian, pancreatic, liver, and gastric cancer [56],
and due to MKP-1 expression, the lung and ovarian cell lines are resistant to
chemotherapy such as cisplatin [57]. TTP mRNA and protein levels are significantly
decreased in tumors of the thyroid, lung, ovary, uterus, and breast compared to
non-transformed tissues [58,59]. Another study showed that the mRNA expression
levels of TTP family proteins were repressed in lung and ovarian cancers [60]. However,
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rare studies clearly elucidate the expression and function of three TTP family members
in a cancer cell. Combining previous researches with our observations, the possible
explanation of MKP-1 overexpression is losing of ZFP36L1 and ZPL36L2 in cancer
cells, and the detailed mechanism can be further investigated.
The abnormal expression of COX-2 in breast cancer cells is reported in many
studies[61,62], and it may involve in tumorigenesis and angiogenesis in the breast
cancers [63,64]. The relationship between COX-2 and TTP is confirmed [65], but
whether the overexpression of COX-2 is regulated by ZFP36L2 in breast cancer cells is
unclear.
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