Proc

Proc. synergistic effect on the expression of the cytokines interleukin (IL)-8, IL-6, and growth-related oncogene (Gro-). By using E-selectin cross-linking and beads coated with CD44 immunopurified from STING agonist-1 Lu1205 cells, we showed that CD44/selectin ligation was responsible for the ICAM-1 up-regulation on HUVECs. Protein kinase C (PKC-) activation was found to be the downstream target of the CD44/selectin-initiated signaling, as ICAM-1 elevation was inhibited by siRNA targeting PKC or a dominant negative form of PKC (PKC DN). Western blot analysis and electrophoretic mobility shift assays (EMSAs) showed that TCCEC contact mediated p38 phosphorylation and binding of the transcription factor SP-1 to its regulation site. In conclusion, CD44/selectin binding signals ICAM-1 up-regulation on the EC surface through a PKCCp38CSP-1 pathway, which further enhances melanoma cell adhesion to ECs during metastasis.Zhang, P., Goodrich, C., Fu, C., Dong, C. Melanoma upregulates ICAM-1 expression on ECs through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCCp38CSP-1 pathway. an increased expression of ICAM-1 (23,C25). In concert with NF-B, several signaling molecules, including c-Jun N-terminal kinase (JNK), p38, IB kinase (IKK), and Src, were found to be involved in soluble factorCinduced ICAM-1 expression (23,C29). The ICAM-1 promoter contains a variety of transcription factorCbinding regions for NF-B, C/EBP, SP-1, and P91 (30, 31). Protein kinase C (PKC) is a family of serine/threonine kinases that mediate IgM Isotype Control antibody (PE) intracellular signaling and can be classified into 3 groups on the basis of their regulatory domains: diacylglycerol- and calcium-regulated, conventional PKC (including PKC-, -I, -II, and -); calcium-independent novel PKC (including PKC-, -, -, -, and -); and diacylglycerol- and calcium-independent, atypical PKC (including PKC- and -) (32). PKC can be activated and bound to cell membranes on phosphorylation (33). PKC, -, and – are known to regulate p38 phosphorylation and ICAM-1 expression in response to stimuli (23,C25, 34). Although those studies outlined some mechanisms by which cell receptor engagement and cytokine stimulation initiate a multifunctional signaling pathway in activated ECs, how flow could modulate these signaling events, especially in the presence of TC adhesion, remains elusive. In the current study, we designed novel adjacent bilayer contact and flow coculture systems to study the effect of direct interactions between TCs and ECs on the regulation of adhesion molecule expression on ECs. We report that melanoma cells possess CD44, which functions as a high-affinity E-selectin ligand to regulate ICAM-1 expression on the endothelium in flow. IL-6 released into the coculture medium primed cell surface E-selectin, which triggered CD44-bindingCinitiated intracellular signaling transduction. By using pharmacological and genetic interventions, as well as a F?rster resonance energy transfer (FRET)-based C kinase STING agonist-1 activation reporter (CKAR), we provided compelling evidence that mechanical signals transmitted from CD44/E-selectin bonds orchestrate the PKC-p38 signaling cascade, thereby increasing the ability of SP-1 to activate ICAM-1 gene transcription. In view of the importance of ICAM-1 in regulating stable adhesion of leukocytes and TCs to the endothelium, our findings shed light on the role of mechanotransduction in the tumor microenvironment and provide insights that may aid in the development of intervention strategies to control the hematogenous dissemination of TCs. MATERIALS AND METHODS Reagents Mouse IgG anti-human E-selectin, CD44H, VCAM-1, and ICAM-1 (clone BBIG-I1) were purchased from R&D Systems (Minneapolis, MN, USA). AffiniPure F(ab)2 fragment goat anti-human IgG (H+L) was from Jackson ImmunoResearch (West Grove, PA, USA). keratanase I, chondroitinase ABC, neuraminidase 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione hydrochloride, G?6976, ammonium pyrrolidine dithiocarbamate (PDTC), mithramycin A, and SB203580 were from Sigma-Aldrich (St. Louis, MO, USA). Anti-phospho-p38 (Thr180/Tyr182), anti-phospho-PKC/II (Thr638/641), anti–tubulin, anti-p38, anti-PKC, and 12-and height for 5 min and then stored at ?80C until used for ELISA. To measure individual cytokine concentrations, we coated each well in a 96-well plate with mouse anti-human capture antibodies diluted in NaHCO3 (pH 8.2) at a final concentration of 2 g/ml (R&D Systems). After the plates were incubated overnight at 4C, each well was blocked for 2 h at room temperature with PBS/1% BSA. Samples and standards were added STING agonist-1 at 100 l/well and incubated overnight at 4C. The wells were incubated.