This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.Y-box protein (YB)-1 of the cold-shock protein family functions in gene transcription and RNA processing. Extracellular functions have not been reported, but the YB-1 staining pattern in inflammatory glomerular diseases, without adherence to cell boundaries, suggests an extracellular occurrence. Here, we show the secretion of YB-1 by mesangial and monocytic cells after inflammatory challenges. It should be noted that YB-1 was secreted through a non-classical mode resembling that of the macrophage migration inhibitory factor. YB-1 release requires ATP-binding cassette transporters, and microvesicles protect YB-1 from protease degradation. Two lysine residues in the YB-1 carboxy-terminal domain are crucial for its release, probably because of post-translational modifications. The addition of purified recombinant YB-1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration. Thus, the non-classically secreted YB-1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

Y-box protein-1 (YB-1) is a known negative regulator of collagen (Col) expression by two different mechanisms, acting directly through binding to an interferon-␥ response element within the col1A2 promoter and/or by physically interacting with p300/ Smad3, thereby abrogating the stimulatory effect of transforming growth factor-␤ (TGF-␤). Here, we report that YB-1 activation via the Jak1 signaling pathway is required and sufficient to confer interferon-␥-dependent activation of the smad7 gene. By binding to a bona fide recognition site within the smad7 promoter, YB-1 up-regulates smad7 transcription, which was additively enhanced by autoinhibitory TGF-␤ signaling. Importantly, the anti-TGF-␤ effect was not only supplied by induced Smad7 expression but was recapitulated in the context of the col1A2 promoter, where YB-1 overexpression abolished the trans-stimulatory TGF-␤ effect in a dominant fashion. In conclusion, YB-1 is the main target of interferon-␥ signaling via Jak1 that exerts antifibrotic action by both interference with TGF-␤ signaling and direct down-regulation of collagen expression.Y-box proteins belong to a family of DNA-and RNA-binding factors, also named cold shock proteins, that are highly conserved during evolution and have been shown to function as regulators of gene transcription and translation (1, 2). A wide range of nucleic acid structures are reported to be specifically bound by Y-box proteins, most of which harbor an inverted CCAAT-box (ATTGG) as the core binding site. YB-1 3 was originally identified by an expression cloning strategy using the Y-box of the major histocompatibility complex class II and epidermal growth factor receptor promoters as probes (3,4). It is a ubiquitously expressed early response gene that is induced among others following interleukin-2 stimulation of cloned T helper lymphocytes (5) and after partial hepatectomy (6). YB-1 has been implicated in the regulation of proliferation-associated genes (e.g. thymidine kinase, proliferating cellular nuclear antigen, and epidermal growth factor receptor (7)), and most recently a direct effect on p53 protein expression and interaction with p53 has been described (8). In regard to ECM synthesis and deposition, YB-1 was identified as a negative regulator in glomerular mesangial cells, where it trans-activates transcription of the matrix metalloproteinase-2 gene via combinatorial interactions with p53 and AP-2, which may be antagonized by nonmetastasizing protein 23 (Nm23) (9). In human embryonic kidney cells and dermal fibroblasts, a repressive function of YB-1 on the col1A2 gene promoter was reported, which is mediated by IFN-␥ (10). Screening of a human fibroblast cDNA expression library with a radiolabeled col1A2 IFN-␥ response element (IgRE) probe exclusively yielded clones with a sequence identical to YB-1 (10). Studies with a mutated IgRE and expression vectors containing partially deleted YB-1 cDNAs that were GFP-tagged for detection by immunofluorescence microscopy indicated that IFN-␥ facilitates nuclear translocation ...

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As eukaryotic cells progress through the cell cycle, multiple genes involved in DNA replication and nucleotide metabolism are coordinately up-regulated shortly before or at the onset of DNA synthesis. These genes among others encompass histones, dihydrofolate reductase, thymidine kinase and synthase, proliferating cell nuclear antigen, topoisomerase II␣, and DNA polymerase ␣ (DPA) 1 /primase (1-3). A concordant up-regulation of the transcription factor Y-box protein 1 (YB-1) with topoisomerase II␣ and proliferating cell nuclear antigen has been previously reported (4); however, a direct involvement of YB-1 in the transcriptional control of the aforementioned genes has not been investigated. DPA is a key component of the chromosomal replication apparatus and is regarded as the principal polymerase involved in eukaryotic DNA replication (5). A role for DPA primase has been found in the checkpoint that couples S phase to mitosis (6). Furthermore, Wahl et al. (7) demonstrated a significant up-regulation of DPA gene transcription during the activation of quiescent (G 0 phase) to proliferating cells (G 1 /S phases). Steady state DPA mRNA levels, synthesis rates of nascent polymerase protein, and enzymatic activity all exhibit a substantial increase before the peak of in vivo DNA synthesis. The concerted increase of these three parameters is consistent with the regulation of this key DNA replication enzyme to a considerable extent at the transcriptional level. Studies performed by Wang and co-workers (7,8) demonstrated that in serum-deprived cells, DPA mRNA, protein, and in vitro activity levels are low, whereas serum addition leads to a coordinate increase in parallel with the onset of DNA synthesis. Prior analyses of the GC-rich TATA-less DPA promoter sequence for cis-acting elements identified a serum response element that is activated in NIH 3T3 cells (8). This element was mapped to sequences Ϫ65/Ϫ17 relative to the transcriptional start site. The 28-bp sequence Ϫ45/Ϫ17 includes an inverted CCAAT box and enhances transcription 10-fold in cycling cells when compared with the minimal activity construct Ϫ17/ϩ45 (8). Specific binding activities that trans-activate DPA gene transcription via this element include CTF1 (9) and CTF/NF-I (10). The importance of this sequence for DPA gene expression has also been demonstrated in the course of human cytomegalovirus infection. Human cytomegalovirus immediate-early protein 1 directly interacts with CTF1 and synergistically trans-activates DPA gene transcription via the inverted CCAAT box (9). Inverted CCAAT boxes also constitute binding sites for Ybox-binding proteins (11). YB-1 binds to DNA as well as RNA in a sequence-specific fashion and is implicated in the transcriptional regulation of a variety of genes (12). Depending on the cellular context, YB-1 may act either as a transcriptional activator or repressor, even of the same gene (13).Close inspection of the DPA gene sequences Ϫ45/Ϫ17 revealed the presence of an inverted CCAAT box on the opposite strand with an inverse rep...