The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including ␣ 6 ␤ 1 . Previous studies have shown that CCN1 interaction with integrin ␣ 6 ␤ 1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin ␣ 6 ␤ 1 . In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-␣ 6 and anti-␤ 1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQC-SKS as the critical determinant for mediating ␣ 6 ␤ 1 -dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other ␣ 6 ␤ 1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits ␣ 6 ␤ 1 -dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin ␣ 6 ␤ 1 directly, we perform affinity chromatography and show that integrin ␣ 6 ␤ 1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin ␣ 6 ␤ 1 , providing the basis for the development of peptide mimetics to examine the functional role of ␣ 6 ␤ 1 in angiogenesis.

Summary Anti‐ribosomal phosphoprotein autoantibodies have been shown to be significantly associated with multiple manifestations of systemic lupus erythematosus (SLE). High levels of interleukin‐10 (IL‐10) have been demonstrated to contribute to lupus susceptibility and severity. In this study, we investigated the molecular mechanisms of anti‐ribosomal phosphoprotein monoclonal antibody (anti‐P mAb)‐induced autoimmune responses. Anti‐P mAb promoted IL‐10 overproduction in a dose‐ and time‐dependent manner in both lipopolysaccharide (LPS)‐activated RAW 264.7 cells and primary human macrophages. Anti‐P mAb enhanced phosphorylation of Akt (PKB; protein kinase B), extracellular signal regulated kinase 1/2 (ERK1/2) and c‐Jun NH2‐terminal kinase 1/2 (JNK1/2), while phosphorylation of p38 remained unaltered. Furthermore, anti‐P mAb decreased glycogen synthase kinase 3 (GSK3) activity and reduced the phosphorylation of IκBα in LPS‐activated macrophages. The Syk, phosphatidylinositol 3‐kinase (PI3K), protein kinase C (PKC), JNK and ERK signalling pathways involved in anti‐P mAb‐triggered IL‐10 secretion were also confirmed using various pharmacological inhibitors. In addition, nuclear factor (NF)‐κB had negative regulatory effects on anti‐P mAb‐triggered IL‐10 secretion. Using reporter plasmids containing the nuclear factor binding sites of NF‐κB, cAMP‐enhanced activation protein 1 (AP‐1), serum response element (SRE) or cyclic AMP response element (CRE), treatment of anti‐P mAb led to activation of the corresponding factors that bind to the AP‐1 site, SRE and CRE in the LPS‐activated macrophages. Furthermore, by transfection with reporter plasmids bearing various lengths of the IL‐10 promoter, the AP‐1 binding site, SRE and CRE were shown to be required for anti‐P mAb‐induced effects. Collectively, our results provide a molecular model for anti‐P mAb‐induced IL‐10 overproduction in LPS‐activated macrophages, which may play a role in the pathogenesis of SLE.

Phosphoglycerate kinase 1 (PGK-1) is a multifunctional protein that is involved in the glycolytic pathway and the generation of the angiogenesis inhibitor angiostatin. In a previous study, we showed that the overexpression of full-length PGK-1 in Lewis lung carcinoma (LLC-1) can reduce tumor growth in vivo by downregulation of COX-2 expression. Phosphoglycerate kinase 1 has two functional domains: a catalytic domain (CD); and a nucleotidebinding domain (NBD). To identify the functional domain of PGK-1 responsible for its antitumor effects, we evaluated the tumorigenicity of LLC-1 cells overexpressing full-length PGK-1 (LLC-1 ⁄ PGK), CD (LLC-1 ⁄ CD), and NBD (LLC-1 ⁄ NBD). Although no difference in tumor cell growth was observed in vitro, the tumor invasiveness was reduced in the LLC-1 ⁄ PGK, LLC-1 ⁄ CD, and LLC-1 ⁄ NBD cells compared to parental LLC-1 cells in vivo. In addition, in vivo tumor growth retardation by LLC-1 ⁄ CD and LLC-1 ⁄ NBD cells was observed, similar to that by LLC-1 ⁄ PGK cells. However, the reduced stability of COX-2 mRNA and downregulation of the COX-2 protein and its metabolite, prostaglandin E2, was only found in LLC-1 ⁄ PGK and LLC-1 ⁄ NBD cells. Low levels of COX-2 were also observed in the tumor mass formed by the modified cells when injected into mice. The results indicate that COX-2 suppression by PGK-1 is independent of its catalytic activity. COX-2 targeting by PGK-1 can be attributed to its NBD and is probably a result of the destabilization of COX-2 gene transcripts brought about by the mRNA-binding property of PGK-1. (Cancer Sci 2010; 101: 2411-2416 L ung cancer is the leading cause of carcinoma-related deaths worldwide, and non-small cell lung carcinoma (NSCLC) constitutes 85% of all lung cancers. Despite advances in lung cancer treatment, poor survival rates are commonly seen in patients with late-stage disease.(1) Therefore, early diagnosis and novel therapeutic approaches are urgently required. Previous studies indicated that the blocking of oncogenic signaling cascades that targeted the epidermal growth factor receptor-and COX-2-regulated pathways yielded promising results in specific subsets of lung cancers.(1,2)Phosphoglycerate kinase 1 (PGK-1), an enzyme comprising a common nucleotide-binding domain (NBD) and two unique catalytic domains (CDI and CDII), is an interesting multifunctional protein. (3,4,(5)(6)(7)(8)(9) In addition to being an essential enzyme in the glycolytic pathway, (3) PGK-1 is well known to play an inhibitory role in tumor angiogenesis by facilitating the formation of angiostatin from plasmin.(8) Moreover, PGK-1 can influence DNA replication and repair in the mammalian nucleus and stimulate viral mRNA synthesis in the cytosol.(5,6) The enzyme also serves as an mRNA-binding protein and is implicated in the negative regulation of the stability of urokinase-type plasminogen activator receptor (uPAR) mRNA.(7) The reduction in uPAR expression and cellular motility by overexpression of PGK-1 has been shown in human lung cancer cells.(7) Peptides of PGK-1 isol...