Y705 and S727 are required for the mitochondrial import and transcriptional activities of STAT3, and for regulation of stem cell proliferation

Abstract

The STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity affects their proliferation rates. As a result, we demonstrated that STAT3 import inside mitochondria requires Y705 phosphorylation by Jak, while its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: while the Y705 mutated STAT3 cannot enter mitochondria, the S727 mutation does not affect the import in the organelle and is responsible for STAT3-dependent mitochondrial transcription. Surprisingly, STAT3-dependent increase of mitochondrial transcription seems independent from STAT3 binding to STAT3 responsive elements. Finally, loss of function experiments, with chemical inhibition of the JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia

“…STAT3 transcriptional activity has also been reported to be positively affected by the phosphorylation of Serine 727 (Levy et al ., 2002; Zhang et al ., 1995; Wen et al ., 1995) and this post-translational modification is triggered by MEK/ERK pathway (Gough et al ., 2013). As we recently demonstrated that the MEK/ERK inhibitor PD98059 blocks the pS727 activities of STAT3 (Peron et al ., 2021), we sought to assess whether the inhibition of S727 phosphorylation can affect the responsiveness of larvae to low oxygen tensions. When HRE:mCherry reporter larvae are treated with PD98059 and incubated in low oxygen tensions, we could not observe significant differences in reporter fluorescence compared to hypoxic DMSO-treated larvae, suggesting that the chemical inhibition of S727 phosphorylation does not affect the transcriptional responsiveness of zebrafish to hypoxia (Fig.…”

“…Zebrafish is an in vivo model in which the pathophysiological roles of HIF1α and JAK/STAT signalling have been extensively studied (Santhakumar et al ., 2012; Gerri et al, 2017; Gerri et al ., 2018; Vettori et al ., 2017; Marchi et al ., 2020; Liu et al, 2017; Peron et al ., 2020; Peron et al ., 2021; Dinarello et al ., 2022). Thus, it appeared as a valid platform to analyze the STAT3-HIF1α crosstalk.…”

“…To investigate the requirement of Stat3 in the transcriptional response to hypoxia, we combined hypoxic and pseudohypoxic treatments with chemical or genetic inhibition of Stat3. To inhibit Stat3 signaling we used AG490, which blocks the Jak-mediated Y705 phosphorylation of Stat3 and abrogates its nuclear transcriptional activity (Park et al, 2014; Peron et al, 2020; Peron et al ., 2021). Larvae treated with this compound were also characterized by a reduction of stat3 gene expression (Fig.…”

“…3B). To further elucidate the role of Serine or Tyrosine phosphorylation in Stat3-dependent regulation of hypoxia transcription, we injected Stat3 mRNAs (that we recently used and validated in Peron et al ., 2021) in double transgenic eggs obtained from the breeding between Tg(7xStat3-Hsv.Ul23:EGFP) ia28 and HRE:mCherry transgenic animals to monitor at the same time the Stat3- and the hypoxia-dependent transcription. Of note, wild type Stat3 mRNA injection upregulated mCherry fluorescence suggesting that the overexpression of Stat3 increases hypoxia-dependent transcription, while Stat3 Y705F mRNA (that encodes a STAT3 that cannot be phosphorylated in 705 position, as described in Minami et al ., 1996) cannot induce the fluorescence of both reporters (Fig.…”

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription

“…No significant differences in reporter fluorescence were detected, meaning that the inhibition of Stat3 does not affect GC/GR-dependent transcription. To confirm this result, we also measured the fluorescence of Tg(9xGCRE-Hsv.Ul23:EGFP) ia20 in stat3 mutant background, taking advantage of a stat3 ia23 zebrafish mutant line [ 51 , 52 ]. No significant differences between stat3 +/+ , stat3 +/ia23 and stat3 ia23/ia23 6 dpf larvae were observed ( Figure S3B ).…”