Evidence that functional interactions of CREB and SF-1 mediate hormone regulated expression of the aromatase gene in granulosa cells and constitutive expression in R2C cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.     

Sorting Nexin 31 Binds Multiple β Integrin Cytoplasmic Domains and Regulates β1 Integrin Surface Levels and Stability

Trafficking of α5β1 integrin to lysosomes and its subsequent degradation is influenced by ligand occupancy and the binding of SNX17 via its protein 4.1, ezrin, radixin, moesin (FERM) domain to the membrane-distal NPxY motif in the cytoplasmic domain of β1 integrin in early endosomes. Two other sorting nexin (SNX) family members, namely SNX27 and SNX31, share with SNX17 next to their obligate phox domain a FERM domain, which may enable them to bind β integrin tails. Here we report that, in addition to SNX17, SNX31 but not SNX27 binds several β integrin tails in early endosomes in a PI3 (phosphatidylinositide 3)-kinase-dependent manner. Similarly like SNX17, binding of SNX31 with β1 integrin tails in early endosomes occurs between the FERM domain and the membrane-distal NPxY motif in the β1 integrin cytoplasmic domain. Furthermore, expression of SNX31 rescues β1 integrin surface levels and stability in SNX17-depleted cells. In contrast to SNX17, expression of SNX31 is restricted and found highly expressed in bladder and melanoma tissue. Altogether, these results demonstrate that SNX31 is an endosomal regulator of β integrins with a restricted expression pattern.     

IGF2 derived from SH-SY5Y neuroblastoma cells induces the osteoclastogenesis of human monocytic precursors

The insulin-like growth factors 2 (IGF2) is a peptide hormone that binds to the insulin-like growth factor 1 receptor (IGF1R) and is abundantly stored in bone. IGF1R is deeply involved in the pathogenesis of many cancers that growth within bone and is also involved in osteoclast biology. Among different cell lines representative of osteolytic tumors, we found a very high expression of IGF2 in SH-SY5Y cells derived from neuroblastoma (NB). We previously showed that NB cells induce an osteolytic process through the Osteoprotegerin/RANKL/RANK and the canonical Wnt pathway system. Here, we hypothesized that NB promotes osteoclastogenesis also via IGF2. First, we demonstrated the presence of IGF1R on the osteoclast basolateral membrane, and we observed a cyclic IGF1R activation along with the differentiation process, also when induced by SH-SY5Y. Moreover, we found that IGF2 mRNA expression in SH-SY5Y cells was further increased when co-cultured with mesenchymal stromal cells, suggesting that IGF2 is important for NB interaction with the bone microenvironment. Finally, the treatment of SH-SY5Y cells with an anti-IGF2 siRNA or the addition of anti-IGF1R molecules impaired NB-induced osteoclastogenesis, even though the chemoattraction of monocytes by NB cells was unaffected. Our findings suggest that in IGF2-producing osteolytic tumors IGF1R is a good candidate for targeted therapies in combination with conventional drugs.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for Toll-like receptor activation and cellular signaling

The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The α-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane Gα(i) subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR Gα(i)-signaling proteins blocks LPS-induced Neu1 activity and NFκB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFκB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling.     

Spy1 induces de-ubiquitinating of RIP1 arrest and confers glioblastoma's resistance to tumor necrosis factor (TNF-α)-induced apoptosis through suppressing the association of CLIPR-59 and CYLD

Glioblastoma multiforme (GBM), a grade-IV glioma, is resistant to TNF-α induced apoptosis. CLIPR-59 modulates ubiquitination of RIP1, thus promoting Caspase-8 activation to induce apoptosis by TNF-α. Here we reported that CLIPR-59 was down-regulated in GBM cells and high-grade glioma tumor samples, which was associated with decreased cancer-free survival. In GBM cells, CLIPR-59 interacts with Spy1, resulting in its decreased association with CYLD, a de-ubiquitinating enzyme. Moreover, experimental reduction of Spy1 levels decreased GBM cells viability, while increased the lysine-63-dependent de-ubiquitinating activity of RIP1 via enhancing the binding ability of CLIPR-59 and CYLD in GBM, thus promoting Caspase-8 and Caspase-3 activation to induce apoptosis by TNF-α. These findings have identified a novel Spy1-CLIPR-59 interplay in GBM cell''s resistance to TNF-α-induced apoptosis revealing a potential target in the intervention of malignant brain tumors.     

Neurogenin 3 recruits CBP co-activator to facilitate histone H3/H4 acetylation in the target gene INSM1

INSM1 is a downstream target gene of neurogenin 3 (ngn3). A promoter construct containing the -426/+40bp region transiently co-transfected into NIH-3T3 cells with a ngn3 expression plasmid resulted in a 12-fold increase in promoter activity. The ngn3/E47 heterodimer selectively binds and activates the E-box3 of the INSM1 promoter. The endogenous ngn3 and CREB-binding protein (CBP) co-activator occupy the INSM1 promoter, resulting in hyper-acetylation of histone H3/H4 chromatin in a human neuroblastoma cell line, IMR-32. Additionally, adenoviral ngn3 can induce endogenous INSM-1 expression in pancreatic ductal carcinoma-1 cells through the recruitment of CBP to the INSM1 promoter and increase the acetylation of the INSM1 promoter region.