Hiroyuki Kunimoto

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.

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.

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.

Laminin-511, inducer of hair growth, is down-regulated and its suppressor in hair growth, laminin-332 up-regulated in chemotherapy-induced alopecia

BACKGROUND Chemotherapy-induced alopecia (CIA) has a devastating cosmetic effect, especially in the young. Recent data indicate that two major basement membrane components (laminin-332 and -511) of the skin have opposing effects on hair growth. OBJECTIVE In this study, we examined the role and localization of laminin-332 and -511 in CIA. METHODS We examined the expression of laminin-332 and -511 during the dystrophic catagen form of CIA induced in C57BL/6 mice by cyclophosphamide (CYP) treatment. RESULTS Our data indicate that both laminin-332 and its receptor alpha 6 beta 4 integrin are up-regulated (both quantitatively and spatially) after mid to late dystrophic catagen around the outer root sheath (ORS) in the lower third of hair follicles in CIA. This up-regulation also occurs at the transcriptional level. In contrast, laminin-511 is down-regulated after mid dystrophic catagen at the protein level, with transcriptional inactivation of laminin-511 occurring transiently at the early dystrophic catagen stage in both epidermal and ORS keratinocytes. Laminin-511 expression correlates with expression of alpha 3 integrin in CIA and we also demonstrate that laminin-511 can up-regulate the activity of the alpha 3 integrin promoter in cultured keratinocytes. Injection of a laminin-511 rich protein extract, but not recombinant laminin-332, in the back skin of mice delays hair loss in CYP-induced CIA. CONCLUSIONS We propose that abrupt hair loss in CIA is, at least in part, caused by down-regulation of laminin-511 and up-regulation of laminin-332 at the transcriptional and translational levels.

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.