3.6. The change of AMPK phosphorylation and the expression of
SP1 and PGAM5 were recovered in Empa treated HK2 cells.
We investigated the change of
phosphorylated AMPK, PGAM5 and SP1
protein in vitro. Similarly, we found a remarkable downregulation of
phosphorylated AMPK and a significant upregulation of PGAM5 and SP1
expression in HK2 cells subjected to HG, and the Empa recovered these
changes significantly (Fig. 4A, F). In order to detect the causality
between the AMPK phosphorylation and the SP1-PGAM5 changes, we
introduced AMPK activator (AICAR)
and inhibitor (Compound C) into
our experiments. We found that both HG and Compound C exposure result in
decreased phosphorylated AMPK,
DRP1S637 and increased SP1 and PGAM5 expression. Both the Empa and the
AICAR could alleviate the changes of AMPK, DRP1S637, SP1 and PGAM5 in
HK2 cells (Fig. 4C, D, E).
Meanwhile, this alleviation effect of Empa diminished again when the
Compound C was administrated (Fig. 4C, D, E).
In order to consolidate the involvement of SP1 and PGAM5 in the AMPK
regulated mitochondrial fission, we examined the fission related protein
in HG treated HK2 cells that subjected to si-SP1 or si-PGAM5. We found
that both
si-PGAM5
and si-SP1 significantly reversed the decrease of DRP1S637 in HK2 cells
subjected to HG (Fig. 4B, H), but
neither si-PGAM5 or si-SP1 could affect the
AMPK phosphorylation (Fig. 4B, G).
Meanwhile, the si-SP1 obviously reduced the PGAM5 expression but the
si-PGAM5 had no effect on SP1 (Fig. 4B, G). The Empa could not only
upregulate the phosphorylated AMPK and DRP1S637, but also decrease the
expression of SP1 and PGAM5 in HG treated HK2 cells (Fig. 4B, G, H).
3.7. The SP1
could bind to the promotor region of PGAM5 and increased its
transcription in HK2 cells.
Finally, we further testify our deduction that the SP1 could bind to the
PGAM5 promoter region directly.
The predicted binding sites within
the SP1 sequence are illustrated in Figure 5A. JASPER analysis revealed
3 putative SP1 binding motifs within the promoter of PGAM5 that located
at 713, 539, 320 bp upstream of the transcription start
(Fig. 5B). We found that
overexpression of SP1
significantly increased the protein levels of PGAM5 in HK2 cells (Fig.
5C, D), which confirmed that SP1 can activate the expression of PGAM5.
As shown in Fig. 5E, the WT
promoter construct of PGAM5 had transcriptional activity in HK2 cells as
compared to the promoterless
pEZX-PG02. The SP1 remarkably increased the transcriptional activity of
the WT promoter. Moreover, the mutant of the SP1
binding site at -539 nt
significantly decreased the transcriptional activity of the PGAM5
promoter. By contrast, the mutant of the SP1 binding site at either -713
or -320 nt did not alter the promoter activity
significantly (Fig.
5E). We next testified the
physical interacts between SP1 and PGAM5 promoter
by chromatin immunoprecipitation
(ChIP) assay using HK2 cells transfected with either SP1 expression
construct or its empty vector. DNA from the immunoprecipitates was then
amplified by PCR with primers located in the promoter region near the
putative SP1 binding site. SP1 was found to physically bind to this
region, whereas no binding was detected in these samples with the
negative control (NC) primers located far from the -539 nt region (Fig.
5F).