Abstract P1071: Concurrent Stat1 And Stat3 Signaling Modulates Tlr4/nfkb-mediated Immunomodulatory And Antifibrotic Effects Of Cardiomyocyte-damaged Released S100a1 Protein In Cardiac Fibroblasts

Abstract

Background: We previously showed that S100A1 released from damaged cardiomyocytes can accelerate post-myocardial infarction in vivo healing. The action of S100A1 as a damage-associated molecular pattern seemed to occur via an immunomodulatory (e.g., IL10 increase) and antifibrotic impact (e.g., COL1A1 and ACTA2 downregulation) in cardiac fibroblasts (CFs) via a TRL4/NKFB pathway. The signaling network in CFs conveying this effect has not been characterized yet. Methods and Results: We performed a 48h time-resolved transcriptome analysis on cultured adult rat CF stimulated with rec. human S100A1 protein (10 -6 M). Bioinformatic GSEA yielded an instant and almost exclusive significant upregulation of the chemokine activity GO-term with a delayed suppression of ECM genes. A transcription factor enrichment analysis for the differentially regulated transcripts indicated an immediate activation of NFKB/RELA with a concurrent temporary STAT1 but lasting STAT3 signaling. A secretome LC-MS/MS-based analysis confirmed the shift of S100A1-treated CFs to mostly chemokine secreting cells. With CCL2 as most secreted chemokine and COL1A1 and ACTA2 as established antifibrotic marker genes for released S100A1, we used a RT-PCR readout for these genes in response to siRNA-based knock-down (KD) of STAT1 and STAT3 in S100A1-treated CFs, while TLR4 KD was used to abrogate NFKB/RELA signaling due to known mechanisms (n=10 each group). S100A1 treatment upregulated CCL2 (16-fold*) and downregulated COL1A1 (3.1-fold*) and ACTA2 (2.9-fold*) mRNA expression vs control (*P<0.01) that was blunted by TLR4 KD. STAT3 KD doubled the CCL2 mRNA increase (32.9-fold*), attenuated COL1A1 mRNA downregulation by S100A1 treatment to 2-fold* but left ACTA2 downregulation unaffected vs contr. (*P<0.05). Conversely, STAT1 KD completely blocked the S100A1 mediated downregulation of ACTA2 but neither impacted S100A1-mediated changes in CCL2 nor COL1A1. Conclusion: Our study identifies concurrent STAT1 and STAT3 activation as a modulator of S100A1-mediated TLR4-NFKB signaling in CFs. As S100A1-activated CFs express e.g., IL-10, we speculate that IL-gp130 trans signaling may convey this effect guiding further studies on S100A1’s extracellular effects in the diseased heart.