Abstract 115: S100A6 Regulates Endothelial Cell Cycle Progression via Modulation of the STAT1 Signaling Pathway

S100A6, also known as Calcyclin, is a 10.5kDa Ca 2+ -binding protein that belongs to the S100 protein family. We have found S100A6 to be highly expressed throughout all vascular layers during the remodeling process in pig coronary arteries after stenting and rat carotid arteries post balloon dilatation injury. It was the objective of this study to decipher S100A6’s function in vascular cells. Abundant S100A6 expression was confirmed in vitro in human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (SMC). Upon serum stimulation, a significant increase of S100A6 protein levels was observed in both cell types. S100A6 depletion due to siRNA transfection lead to a profoundly reduced proliferation rate in response to growth factors (e.g. VEGF-A), shown by stagnating cell counts and decreased EdU incorporation in HUVEC and SMC. Of note, S100A6 depletion caused a significant migration deficit of SMC. Also, reduced S100A6 levels in HUVEC lead to an increase in cellular senescence, as measured by the expression of senescence associated β-galactosidase expression. To decipher the molecular mechanisms that lead to the phenotype of S100A6 depleted vascular cells, a time-resolved gene expression analysis was carried out in HUVECs and revealed S100A6 to be in control of antiproliferative signaling pathways, while typical pro-proliferative signaling, e.g. the MAPK pathway, was not disrupted. S100A6 depletion caused increased expression and activation of the STAT1 signaling pathway. With increased STAT1 activity, interferon-inducible protein (e.g. IFITM1) expression was upregulated and lead to stabilization and increased expression of the p53 pathway, specifically p21. We propose that S100A6, found to co-localize with the VEGFR2 in a Ca 2+ -dependent manner, controls antiproliferative signaling by inhibition of the STAT1-phosphorylation by the VEGFR2 and suppression of the STAT1-p53 signaling cascade in human endothelial cells. Endothelialization is a hallmark of vascular healing after stenting and balloon dilatation, for example, while it is of utmost importance to block neointima formation. Understanding these signaling pathways is critical to better direct optimization of interventional therapies.

Thrombopoietin receptor down-modulation by JAK2 V617F: restoration of receptor levels by inhibitors of pathologic JAK2 signaling and of proteasomes

The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.

Transforming growth factor-β1 and activin A generate antiproliferative signaling in thyroid cancer cells

Transforming growth factor-beta 1 (TGF-β1) and activin A (ActA) induce similar intracellular signaling mediated by the mothers against decapentaplegic homolog (SMAD) proteins. TGF-β1 is a potent antimitogenic factor for thyroid follicular cells, while the role of ActA is not clear. In our study, the proliferation of TPC-1, the papillary thyroid carcinoma cell line, was reduced by both recombinant ActA and TGF-β1. Due to the concomitant expression of TGF-β1 and ActA in thyroid tumors, we investigated the effects of either TGF-β1 or ActA gene silencing by RNA interference in TPC-1 cells in order to distinguish the specific participation of each in proliferation and intracellular signaling. An increased proliferation and reduced SMAD2, SMAD3, and SMAD4 mRNA expression were observed in both TGF-β1 and ActA knockdown cells. Recombinant TGF-β1 and ActA increased the expression of inhibitory SMAD7, whereas they reduced c-MYC. Accordingly, we detected a reduction in SMAD7 expression in knockdown cells while, unexpectedly, c-MYC was reduced. Our data indicate that both TGF-β1 and ActA generate SMADs signaling with each regulating the expression of their target genes, SMAD7 and c-MYC. Furthermore, TGF-β1 and ActA have an antiproliferative effect on thyroid papillary carcinoma cell, exerting an important role in the control of thyroid tumorigenesis.