We report here that the PP1 inhibitor, calyculin A, caused a marked increase in phosphorylation of STAT3 on S727, which had been previously linked to enhanced gene expression by STAT3 at certain promoters (Zhang and others 1995
; Schuringa and others 2001
; Ray and others 2002
; Kurdi and Booz 2007
). Okadaic acid and fostriecin, which exhibit almost an exclusively inhibitory effect on PP2A, did not cause a similar increase in STAT3 S727 phosphorylation, thus showing that PP1 or PP1-related protein is the phosphatase responsible for STAT3 S727 dephosphorylation in HMEC-1. Although several studies have implicated PP2A in dephosphorylating STAT3 S727 (Liang and others 1999
; Woetmann and other 1999
; Zhang and others 2002a
; Togi and others 2009
), a recent study found evidence that PP1 and not PP2A is responsible for STAT3 S727 dephosphorylation in a variety of human tumor cell lines (Haridas and others 2009
The role of STAT3 S727 that lies within the transcription activation domain is complicated and best described as myriad. Evidence has been reported supporting the conclusion that phosphorylation of this residue regulates STAT3 subcellular distribution (Woetmann and others 1999
), tyrosine phosphorylation (possibly through recruitment of a phosphatase) (Zhang and others 2002a
; Booz and others 2003
), cofactor recruitment (e.g., histone deacetylase p300 and Sp1) (Schuringa and others 2001
; Yang and others 2005
), maximal transcriptional activity (Zhang and others 1995
), receptor binding (Zhang and others 2002b
), and homodimerization (Zhang and others 1995
Based on what has been reported, one might expect that simultaneous Y705 and S727 phosphorylation would enhance STAT3-related gene expression. However, we observed just the opposite, which was due in part to the inhibition and degradation of p300/CBP seen with calyculin A. Most likely, these actions occurred indirectly as the result of enhanced (stress) kinase signaling that would occur with inhibition of the repressive phosphatase actions of PP1 on these signaling pathways. Factors that control the stability of p300/CBP are poorly understood. In cardiac myocytes, the anticancer agent doxorubicin was found to cause p300 hyperphosphorylation and degradation via the p38 mitogen-activated protein kinases (Poizat and others 2005
). Protein kinase Cδ, AMP-activated protein kinase, and salt inducible kinase 2 were reported to phosphorylate p300 on S89, thereby attenuating its ability to function as an acetylase (Yang and others 2001
; Yuan and others 2002
; Bricambert and others 2010
). Whether p300/CBP is a direct target of PP1 is a question for future investigation.
Inhibition of DNA binding also partially explains the effect of calyculin A on STAT3-related gene expression. We observed that treatment with calyculin A alone strongly reduced binding of nuclear extracts to a STAT3 consensus binding site (), although nuclear levels of STAT3 were not affected (). Others reported that calyculin A reduced binding of STAT3 from cutaneous T lymphoma cells to various STAT3 binding elements (Woetmann and others 1999
). The explanation suggested was that increased serine phosphorylation of STAT3 caused decreased STAT3 tyrosine phosphorylation and consequently reduced STAT3 DNA binding. However, we did not observe a marked decrease in STAT3 phosphorylated on Y705 in response to calyculin A (). An alternative explanation may be increased phosphorylation of STAT3 at 2 other sites, namely S691 or T714, which are known to be affected by DNA damage or the cell cycle, respectively. In fact, calyculin A was reported to cause increased threonine phosphorylation of STAT3 (Woetmann and others 1999
). Although S691 and T714 phosphorylation likely have physiological relevance, to the best of our knowledge, the effect of phosphorylation of these sites on STAT3 DNA binding has not been reported and warrants investigation. In any case, the results shown in indicate that the positive effect of STAT3 Y705 phosphorylation on DNA binding can offset any potential diminution in DNA binding produced by phosphorylation at another site.
LIF-induced STAT3 nuclear levels at 60
min tended to be higher in the absence of calyculin A (). However, the increase did not reach statistical significance. Although STAT3 was originally identified as a latent cytoplasmic transcription factor that translocates to the nucleus on cytokine-induced Y705 phosphorylation, STAT3 is now known to constitutively shuttle between the cytoplasm and nucleus independent of its phosphorylation state (Sehgal 2008
). Thus, subcellular distribution is unlikely to explain our findings on impaired LIF-induced SOCS3
gene expression on cotreatment with calyculin A (). Both SOCS3
contain a STAF_HOXF_STAT framework within their promoters, and their induction is commonly associated with an inflammatory state. SOCS3 acts as a classic negative inhibitor of JAK-STAT signaling, whereas CCL2 is important for the recruitments of lymphocytes to sites of tissue injury and inflammation. Our results raise the possibility that expression of CCL2 and SOCS3 may be tamped or kept under control with increasing stress by attenuation of PP1 activity.
Although we demonstrated that oxidative stress mimicked the effects of calyculin A on STAT3 and p300 phosphorylation, other stress stimuli may be involved as well. Notably, PP1 inhibition by calyculin A in lung microvascular cells has been linked to disruption of barrier function, thus suggesting a role for this phosphatase in cell architecture (Kelly and others 1998
). Consistent with that possibility, we observed that HMEC-1 seemed to adopt a more rounded appearance in response to treatment with calyculin A (unpublished observation). The regulatory mechanism described here may come into play in attenuating certain aspects of IL-6 type cytokine inflammatory signaling when other stresses are present in the cell.
In conclusion, our findings highlight the pivotal role of a serine threonine phosphatase that is most likely PP1 in controlling the nuclear actions of STAT3 by balancing opposing signaling processes (). On one hand, PP1 exerts positive actions on the transcriptional activity of STAT3 by preventing p300/CBP phosphorylation and degradation, and potentially opposing phosphorylation of STAT3 at 1 or 2 inhibitory sites. On the other hand, PP1 serves to attenuate STAT3 transcriptional activity by dephosphorylating the site responsible for p300/CBP recruitment. p300/CBP enhances STAT3 transcriptional activity by allowing for the acetylation of histone at certain promoters containing STAT3 responsive elements (Ray and others 2002
; Ray and others 2008
). Understanding how these opposing signaling events are coordinated and affected by stress could lead to novel therapeutic strategies to control the impact of inflammation on microvascular endothelial cells.
FIG. 9. Scheme illustrating the pivotal role of PP1 in controlling the actions of STAT3 on gene expression in HMEC-1 by exerting both positive and negative actions. STAT3 transcriptional activation by the IL-6 type cytokines is due to enhanced phosphorylation (more ...)