S100A4, a member of the S100 family of proteins, plays an important role in matrix remodeling by up-regulating the expression of matrix metalloproteinases (MMPs). We have previously shown that S100A4 is overexpressed in diseased cartilage and that extracellular S100A4 stimulates MMP-13 production, a major type II collagen-degrading enzyme, via activation of receptor for advanced glycation end product signaling. In the present study, using human articular chondrocytes, we show that intracellular S100A4 translocated into the nucleus upon interleukin-1 (IL-1) stimulation and translocation required post-translational modification of S100A4 by the sumo-1 protein. Two sumoylation sites were identified on the S100A4 molecule, Lys 22 and Lys 96 . Mutation of these lysine residues abolished the ability of S100A4 to be sumoylated and to translocate into the nucleus. Blocking of sumoylation and nuclear transport of S100A4 inhibited the IL-1-induced production of MMP-13. Nuclear S100A4 was bound to the promoter region of MMP-13 in IL-1-treated cells. Thus, we demonstrate a novel mechanism for sumoylated S100A4 as a regulator of expression of the MMP-13 gene.
This is a response to a letter by Berge et al. (1)The role of S100A4 in the regulation of expression of MMPs is well documented. However the molecular mechanism of this regulation is not clearly understood. In this article, using primary cultures of human chondrocytes, we have presented evidence that S100A4 is sumoylated, translocates to the nucleus, and regulates MMP-13 expression through an interaction with the MMP-13 promoter. We thank the Journal for giving us the opportunity to answer the readers' concerns.Response 1: The intent of the immunocytochemical staining shown in Fig.1A was to examine nuclear translocation of S100A4 after IL-1 stimulation. When the images were obtained we focused on the nuclear staining and took low magnification images in order to better evaluate nuclear staining in as many cells as possible at each time point. This resulted in a fainter appearance of the cytoplasmic staining. The original figure submitted for review and subsequently for the publication did show the cytoplasmic staining for S100A4 at the 0-and 120-min time points although it was faint. When the figure was further processed for publication, the reproduction, which included a reduction in size, makes it difficult to appreciate the cytoplasmic staining. However, the immunoblotting results in Fig.1B support the finding of nuclear translocation of S100A4.Response 2: In Fig.2B we immunoblotted with antibodies for both S100A4 and SUMO in order to detect both sumoylated and unsumoylated S100A4 on the same blot. Therefore, the native (unmodified) S100A4 protein band is seen at the bottom of the blot just below the 15-kDa marker and a slower moving S100A4 (sumo-modified S100A4) at a higher molecular weight. The specificity of the slower migrating S100A4 protein band as sumo-S100A4 in lane 2 was confirmed when the in vitro sumoylation reaction was performed without ATP, a critical component required for the sumoylation process, as shown in lane 1. The figure legend did not make it clear that the blot shown in Fig.2B was incubated with both antibodies, and this may have contributed to the readers' confusion.Response 3: We did not show basal levels here since we have previously published data with the endogenous intracellular levels of S100A4 in chondrocytes. Please refer to our earlier publication (2).Response 4: Because the experiments shown in Fig.4, B and D, were performed with different sets of donor cells at different times it is not possible to directly compare the -fold increase in MMP-13 expression between the panels. This is because, for our experiments, we used primary chondrocytes obtained from tissue donors of different ages and genetic backgrounds, which, unlike cell lines, results in variable levels of response to IL-1. However, experiments within each panel were performed with the cells obtained from the same set of donor tissues and are normalized for the level of MMP-13 expression in the empty vector control so that the data within the panel is comparable. The p value for data points in Fig. 4D: bars 1 vers...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.