Hirotada Kojima

SIRT2 down-regulation in HeLa can induce p53 accumulation via p38 MAPK activation-dependent p300 decrease, eventually leading to apoptosis

We previously reported that sirtuin 2 (SIRT2), a mammalian member of the NAD+‐dependent protein deacetylases, participates in mitotic regulation, specifically, in efficient mitotic cell death caused by the spindle checkpoint. Here, we describe a novel function of SIRT2 that is different from mitotic regulation. SIRT2 down‐regulation using siRNA caused apoptosis in cancer cell lines such as HeLa cells, but not in normal cells. The apoptosis was caused by p53 accumulation, which is mediated by p38 MAPK activation‐dependent degradation of p300 and the subsequent MDM2 degradation. Sirtuin inhibitors are emerging as antitumor drugs, and this function has been ascribed to the inhibition of SIRT1, the most well‐characterized sirtuin that deacetylases p53 to promote cell survival and also binds to other proteins in response to genotoxic stress. This study suggests that SIRT2 can be a novel molecular target for cancer therapy and provides a molecular basis for the efficacy of SIRT2 for future cancer therapy.

The STAT3-IGFBP5 axis is critical for IL-6/gp130-induced premature senescence in human fibroblasts

Cells undergo senescence in response to various conditions, including telomere erosion, oncogene activation and multiple cytokines. One of these cytokines, interleukin-6 (IL‑6), not only functions in the immune system, but also promotes cellular senescence and cancer. Here we demonstrate that IL‑6 and the soluble IL‑6 receptor (sIL‑6R) induce premature senescence in normal human fibroblasts by establishing a senescence-inducing circuit involving the signal transducer and activator of transcription 3 (STAT3) and insulin-like growth factor-binding protein 5 (IGFBP5). Stimulating TIG3 fibroblast cells with IL‑6/sIL‑6R sequentially caused an increase in reactive oxygen species (ROS) as early as day 1, followed by the DNA damage response, p53 accumulation and, finally, senescence on days 8–10. We found that STAT3 was required for the events leading to senescence, including the initial early-phase ROS increase and the induction of IL‑1α/β, IL‑6 and CXCL8 mRNAs 4–5 d after IL‑6/sIL‑6R stimulation, suggesting that STAT3’s role is indirect. We searched for STAT3-downstream molecule(s) responsible for the senescence-inducing activity in the supernatants of stimulated TIG3 and identified IGFBP5 as a major STAT3 mediator, because IGFBP5 was expressed from the early phase through the entire senescence process and was responsible for IL‑6/STAT3-induced ROS increase and premature senescence. Thus, IL‑6/sIL‑6R forms a senescence-inducing circuit involving the STAT3-IGFBP5 axis as a key triggering and reinforcing component.

IL-6-STAT3 signaling and premature senescence.

Cytokines play several roles in developing and/or reinforcing premature cellular senescence of young cells. One such cytokine, interleukin-6 (IL-6), regulates senescence in some systems in addition to its known functions of immune regulation and promotion of tumorigenesis. In this review, we describe recent advances in studies on the roles of IL-6 and its downstream signal transducer and activator of transcription 3 (STAT3) in regulating premature cellular senescence. IL-6/sIL-6Rα stimulation forms a senescence-inducing circuit involving the STAT3-insulin-like growth factor-binding protein 5 (IGFBP5) as a key axis triggering and reinforcing component in human fibroblasts. We describe how cytokines regulate the process of senescence by activating STAT3 in one system and anti-senescence or tumorigenesis in other systems. The roles of other STAT members in premature senescence also will be discussed to show the multiple mechanisms leading to cytokine-induced senescence.

Phospho‐Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho‐Tyr705 largely through TC45

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor. It is activated by cytokines, including interleukin‐6 (IL‐6) through phosphorylation at Tyr705 (pY705), which is required for its dimerization and nuclear translocation. However, the role of Ser727 phosphorylation, occurring during activation, remains poorly understood. Using a combination of HepG2‐stat3‐knockdown cells reconstituted with various STAT3 mutants and protein kinase inhibitors, we showed that phospho‐S727 has an intrinsic mechanism for shortening the duration of STAT3 activity, in turn shortening the duration of socs3 mRNA expression. Both STAT3WT and STAT3Ser727Asp (S727D) but not STAT3Ser727Ala (S727A) showed rapid dephosphorylation of pY705 after the inhibition of tyrosine kinases. We found that the nuclear TC45 phosphatase is most likely responsible for the phospho‐S727‐dependent pY705 dephosphorylation because TC45 knockdown caused prolonged pY705 with sustained socs3 mRNA expression in STAT3WT but not in STAT3S727A, and overexpressed TC45 caused rapid dephosphorylation of pY705 in STAT3WT but not in STAT3S727A. We further showed that phospho‐S727 did not affect the interaction of TC45 with STAT3, and that a reported methylation at K140 of STAT3 occurring after phospho‐S727 was not involved in the pY705 regulation. These findings indicate that phospho‐Ser727 determines the duration of STAT3 activity largely through TC45.

SIRT2 knockdown increases basal autophagy and prevents postslippage death by abnormally prolonging the mitotic arrest that is induced by microtubule inhibitors.

Mitotic catastrophe, a form of cell death that occurs during mitosis and after mitotic slippage to a tetraploid state, plays important roles in the efficacy of cancer cell killing by microtubule inhibitors (MTIs). Prolonged mitotic arrest by the spindle assembly checkpoint is a well‐known requirement for mitotic catastrophe, and thus for conferring sensitivity to MTIs. We previously reported that turning off spindle assembly checkpoint activation after a defined period of time is another requirement for efficient postslippage death from a tetraploid state, and we identified SIRT2, a member of the sirtuin protein family, as a regulator of this process. Here, we investigated whether SIRT2 regulates basal autophagy and whether, in that case, autophagy regulation by SIRT2 is required for postslippage death, by analogy with previous insights into SIRT1 functions in autophagy. We show, by combined knockdown of autophagy genes and SIRT2, that SIRT2 serves this function at least partially by suppressing basal autophagy levels. Notably, increased autophagy induced by rapamycin and mild starvation caused mitotic arrest for an abnormally long period of time in the presence of MTIs, and this was followed by delayed postslippage death, which was also observed in cells with SIRT2 knockdown. These results underscore a causal association among increased autophagy levels, mitotic arrest for an abnormally long period of time after exposure to MTIs, and resistance to MTIs. Although autophagy acts as a tumor suppressor mechanism, this study highlights its negative aspects, as increased autophagy may cause mitotic catastrophe malfunction. Thus, SIRT2 offers a novel target for tumor therapy.

Cytoplasmic c-Fos induced by the YXXQ-derived STAT3 signal requires the co-operative MEK/ERK signal for its nuclear translocation

A STAT3 (signal transducer and activator of transcription 3)‐ and a MEK/Erk‐mediated signal can be activated by cytokines, including IL‐6 (interleukin‐6), PDGF, and EGF. Recently, STAT3 and an ERK‐signal were shown to co‐operatively activate the c‐fos gene. Activation of a truncated form of the IL‐6 receptor subunit, gp130, that had only one YXXQ motif, induced both c‐Fos and JunB in NIH3T3 cells through STAT3 without an apparent increase in the AP‐1 (activator protein‐1) activity. In contrast, concomitant stimulation of the STAT3 signal and a MEK/Erk‐signal markedly increased AP‐1 activity with enhanced c‐Fos expression. Surprisingly, the c‐Fos induced by the YXXQ‐signal alone was localized to the cytoplasm, from which it translocated into the nucleus following TPA (12‐O‐tetradecanoyl‐phorbol 13‐acetate) treatment in a MEK/Erk‐dependent manner. c‐Fos that was expressed from a constitutive promoter localized to the nucleus and did not move into the cytoplasm in response to the YXXQ‐signal. Rather, the YXXQ‐signal was required during c‐Fos production for it to be retained in the cytoplasm. Thus, the YXXQ‐signal induces c‐Fos expression through STAT3 and anchors the new c‐Fos in the cytoplasm. In addition, the YXXQ‐signal and an Erk signal co‐operatively cause c‐Fos activation in the nucleus.

Deacetylation of the mitotic checkpoint protein BubR1 at lysine 250 by SIRT2 and subsequent effects on BubR1 degradation during the prometaphase/anaphase transition

Mitotic catastrophe, a form of cell death that occurs during mitosis and after mitotic slippage to a tetraploid state, plays an important role in the efficacy of cancer cell killing by microtubule inhibitors. Prolonged mitotic arrest at the spindle assembly checkpoint (SAC) is a well-known requirement for mitotic catastrophe and, thus, for conferring sensitivity to microtubule inhibitors. We previously reported that downregulation of SIRT2, a member of the sirtuin family of NAD+-dependent deacetylases, confers resistance to microtubule inhibitors by abnormally prolonging mitotic arrest and thus compromising the cell death pathway after mitotic slippage. Thus, turning off SAC activation after a defined period is an additional requirement for efficient post-slippage death. Here, we investigated whether SIRT2 deacetylates BubR1, which is a core component of the SAC; acetylation of BubR1 at lysine 250 (K250) during prometaphase inhibits its APC/C-dependent proteolysis and thus regulates timing in anaphase entry. We showed that SIRT2 deacetylates BubR1 K250 both in vitro and in vivo. We also found that SIRT2 knockdown leads to increased levels of BubR1 acetylation at prometaphase; however, this increase is not substantial to elevate the levels of total BubR1 or delay the transition from prometaphase to anaphase. The present study shows that SIRT2 is a deacetylase for BubR1 K250, although the abnormally prolonged SAC activation observed in SIRT2 knockdown cells is not accompanied by a change in BubR1 levels or by delayed progression from prometaphase to anaphase.

S1-1/RBM10: Multiplicity and cooperativity of nuclear localisation domains

S1‐1, also called RBM10, is an RNA‐binding protein of 852 residues. An alteration of its activity causes TARP syndrome, a severe X‐linked disorder with pre‐ or post‐natal lethality in affected males. Its molecular function, although still largely unknown, has been suggested to be transcription and alternative splicing. In fact, S1‐1 localises in the nucleus in tissue cells and cultured cells.

Interleukin-6 Induces Premature Senescence Involving Signal Transducer and Activator of Transcription 3 and Insulin-Like Growth Factor-Binding Protein 5

Normal cells undergo senescence in response to telomere erosion, various stresses causing DNA damage, and certain cytokines. One such cytokine, interleukin-6 (IL-6), a multifunctional cytokine, can act on multiple lineages of cells together with soluble IL-6 receptor to induce cell proliferation, differentiation, and even promotion of tumorigenesis. We studied the molecular mechanisms by which IL-6 and soluble IL-6R (sIL-6R) cause premature senescence using primary human TIG3 fibroblasts. Stimulation of TIG3 cells with IL-6/sIL-6R sequentially caused generation of reactive oxygen species (ROS) as early as day 1, followed by DNA damage, p53 accumulation, and finally senescence on days 8–10. Signal transducer and activator of transcription 3 (STAT3) was required for the early and late events leading to senescence, including the early-phase increase of ROS and senescence-associated secretary phenotype (SASP) occurring 4–5 days after IL-6/sIL-6R stimulation. Interestingly, the STAT3 function was indirect, and insulin-like growth factor-binding protein 5 (IGFBP5) secreted into the supernatants was identified as the STAT3-downstream molecule responsible for the IL-6/STAT3-induced ROS generation and premature senescence. IGFBP5 was consistently expressed from the initial phase through the entire senescence process, the profile being quite different from that of SASP. Thus, IL-6/sIL-6R forms a senescence-inducing circuit involving the STAT3–IGFBP5 axis as a key triggering and reinforcing component.