Cell lysates were then subjected to Western blot analysis for the indicated proteins. level decreases cell surface-bound IL-1 level, NF-B activity and SASP production. Moreover, S100A13 overexpression promotes oncogene Ras-induced cell senescence (Ras OIS), Doxorubicin-induced cancer cell senescence (TIS) and replicative senescence, while impairment of non-classical secretory pathway of IL-1 delays cellular senescence. In addition, intervention of S100A13 affects multiple SASP and cellular senescence mediators including p38, -H2AX, and mTORC1. Taken together, our findings unveil a critical role of the non-classical secretory pathway of IL-1 in cellular senescence and SASP regulation. Keywords: S100A13, non-classical protein secretory pathway, IL-1, SASP, Cu2+, cell senescence INTRODUCTION Cellular senescence is a permanent cell cycle arrest state in response to various intracellular and extracellular stimuli such as telomere erosion because of repeated cell division (replicative senescence), DNA damage, oxidative stress, and oncogenes including Ras or Myc activation, etc [1]. One hallmark of senescence is that senescent cells secret multiple pro-inflammatory cytokines, Mouse monoclonal to CRTC2 chemokines, growth factors, and other proteins which is referred to as senescence-associated secretory phenotype (SASP) [1]. C646 The SASP has been shown to have context-dependent pleiotropic biological and physiological functions. For instance, SASP has tumor suppressive roles either via cell autonomous mechanism to reinforce cell senescence [2], or using immune surveillance mechanism via cell non-autonomous fashion [3]. The SASP factors also assist tissue repair, embryonic development, as well as in vivo cell reprogramming through paracrine manner [4C6]. However, the mounting evidences also show that SASP factors can promote tumor growth and invasion, and contribute to many age-related diseases and aging in late-life [7]. Two transcription factors NF-B and C/EBP are required for the SASP genes transcription [2, 8]. The persistent activation of ATM/ATR-CHK1/CHK2-mediated DNA damage response (DDR) pathway [9], and p38 MAPK-mediated stress response pathway [10] are reported to regulate NF-B activity and SASP genes expression independently. Cell surface-bound IL-1 is an upstream regulator of SASP genes expression by feed forward inducing NF-B activity [11]. The DDR-dependent activation of transcription factor GATA4 has also been reported to regulate NF-B activity and SASP genes induction [12]. More recently, it has been shown that the innate immunity cytosolic DNA-sensing cGASCSTING pathway is essential for SASP genes induction by stimulating NF-B activity C646 [13C15]. SASP factors exert their functions via either autocrine or paracrine manner. In general, most SASP factors are secreted to extracellular compartment via classical endoplasmic reticulum (ER)-Golgi protein secretory pathway [16]. C646 However, a minority of proteins without a hydrophobic signal peptide located usually at the N-terminus, secret to cell surface independent of conventional secretory pathway, which is termed as non-classical secretory pathway [17]. IL-1, as a crucial SASP factor, secrets to cell membrane surface via the non-classical secretory pathway [17]. First, S100A13, a member of a large gene family of small acidic proteins [18], binds to IL-1, and constitutes the core component of the multiprotein complex. The combination of these two proteins is the key step in the non-classical secretion of IL-1 [19]. Then, this complex interacts with Cu2+ ions and migrates close to the acidic environment of the inner leaflet of the cell membrane [20, 21]. Last, IL-1 is secreted to cell surface [21]. During cellular senescence, cell surface-bound IL-1 binds to its receptor IL-1R in a juxtacrine fashion to stimulate NF-B activity, thus, IL-1 and NF-B comprise a positive feedback loop and IL-1 acts as an upstream regulator of SASP induction [11]. However, the state of the non-classical secretory pathway of IL-1 during cellular senescence remains unknown, and whether this pathway involves in the SASP induction and cellular senescence has not been defined. In this study, we present that S100A13 and Cu2+, two critical components in mediating the non-classical secretion of IL-1, play crucial roles in modulating NF-B activity and SASP expression, as well as cellular senescent response. RESULTS S100A13 is induced and regulates cell surface-bound IL-1 level during cell senescence To investigate whether S100A13-dependent non-classical secretory pathway of IL-1 participates in regulating SASP expression, we used IMR90 cells expressing ER:Ras fusion protein (ER:Ras-IMR90 cells) as a oncogene Ras-induced cell senescence model (Ras OIS) which developed strong SASP. It is reported that human colon cancer cells HCT116 can be induced to senescence by low concentration of Doxorubicin treatment, and possess typical senescent cell features such as the persistent DDR, the up-regulation of NF-B activity and SASP expression which is similar to replicative senescence and other stimuli-induced premature senescence [22]. Therefore, we took this advantage to use Dox-induced HCT116 cell senescence as.