Response to Berge et al.: Comment on the Importance of S100A4 in Regulation of MMP-13
Year: 2010
Type: article
Abstract: 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-1β 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-1β. 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 versus 4 = 0.05 and bars 2 versus 5 = 0.045.
Response 5: The data presented in panels A and B of Fig. 4 are two different readouts for the effect of IL-1β on MMP-13 expression performed with different sets of donor cells and cannot be directly compared. In Fig. 4A, we are showing the effect of IL-1β on the production of MMP-13 protein in the conditioned media from the cells transfected with S100A4 wt plasmid and various mutants of S100A4, and the data are presented in pg/ml MMP-13. In Fig. 4B, we are measuring the effect of IL-1β on the mRNA expression of MMP-13, and the data are represented as -fold change over pEGFP vector. Fig. 4A, bar 2, is the amount of MMP-13 in the media from cells transfected with S100A4 wild type and stimulated with IL-1 whereas bar 5 in Fig. 4B is the -fold increase in MMP-13 mRNA in cells transfected with the S100A4k96R mutant and stimulated with IL-1β, and this shows a 2.25-fold increase over control, not 4-fold.
Source: Journal of Biological Chemistry
Institution: Wake Forest University
Language: English
Topic: S100 Proteins and Annexins
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2050808924