MT1‐MMP modulates melanoma cell dissemination and metastasis through activation of MMP2 and RAC1

Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1‐MMP is a membrane‐associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion‐promoting, pro‐tumorigenic MMP in cancer. We show that deregulation of MT1‐MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1‐MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1‐MMP in melanoma pathogenesis. We demonstrate that MT1‐MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1‐MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1‐MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1‐MMP is required to sustain RAC1 activity and promote MT1‐MMP‐dependent cell motility. These data highlight a novel MT1‐MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.     

Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low‐density lipoprotein receptor‐related protein 1 through MAPK‐ERK1/2 and NF‐κB activation in macrophage‐derived cell lines

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP‐9) expression and protein secretion through the activation of MAPK‐ERK and NF‐κB signaling pathways. Previously, we demonstrated that activated α₂‐macroglulin (α₂M*) through the interaction with its receptor low‐density lipoprotein receptor‐related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK‐ERK1/2. In the present work, we examined whether α₂M*/LRP1interaction could induce the MMP‐9 production in J774 and Raw264.7 macrophage‐derived cell lines. It was shown that α₂M* promoted MMP‐9 expression and protein secretion by LRP1 in both macrophage‐derived cell lines, which was mediated by the activation of MAPK‐ERK1/2 and NF‐κB. Both intracellular signaling pathways activated by α₂M* were effectively blocked by calphostin‐C, suggesting involvement of PKC. In addition, we demonstrate that α₂M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA‐AM, the α₂M*‐induced MAPK‐ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF‐κB, it was shown that the α₂M*‐induced MMP‐9 protein secretion was inhibited, indicating that the MMP production promoted by the α₂M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607–617, 2010. © 2010 Wiley‐Liss, Inc.     

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3′‐Diindolylmethane (DIM) is a known anti‐tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA‐uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B‐DIM could be mediated via inactivation of uPA‐uPAR system. We found that B‐DIM treatment as well as silencing of uPA‐uPAR led to the inhibition of cell growth and motility of MDA‐MB‐231 cells, which was in part due to inhibition of VEGF and MMP‐9. Moreover, silencing of uPA‐uPAR led to decreased sensitivity of these cells to B‐DIM indicating an important role of uPA‐uPAR in B‐DIM‐mediated inhibition of cell growth and migration. We also found similar effects of B‐DIM on MCF‐7, cells expressing low levels of uPA‐uPAR, which was due to direct down‐regulation of MMP‐9 and VEGF, independent of uPA‐uPAR system. Interestingly, over‐expression of uPA‐uPAR in MCF‐7 cells attenuated the inhibitory effects of B‐DIM. Our results, therefore, suggest that B‐DIM down‐regulates uPA‐uPAR in aggressive breast cancers but in the absence of uPA‐uPAR, B‐DIM can directly inhibit VEGF and MMP‐9 leading to the inhibition of cell growth and migration of breast cancer cells. J. Cell. Biochem. 108: 916–925, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of HIV‐1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction

Background

We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c‐c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T‐lymphocytes.

Methods

In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL‐CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL‐CD34+ cells and in the terminally differentiated macrophages using macrophage colony‐stimulating factor, granulocyte macrophage colony‐stimulating factor, interleukin‐3 and stem cell factor.

Results

Quantitative real‐time polymerase chain reaction for CCR5 mRNA indicated over 90% reduction of CCR5 mRNA levels in CCR5 shRNA‐transduced population. The cells with knockdown of CCR5 expression acquired resistance to R5 tropic HIV‐1 NFN‐SX strain. We also developed a novel approach utilizing a mCherry‐CCR5 chimeric reporter to assess the effectiveness of CCR5 target down‐regulation in macrophages directly. Both the shRNA and the reporter were maintained throughout HPC differentiation to macrophages without apparent cytotoxicity.

Conclusions

The present study demonstrates a novel method to simply and directly assess the function of small interfering RNA and the effective inhibition of HIV‐1 infection by a potential potent shRNA to CCR5 delivered into macrophages derived from HPCs. Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibition of interleukin‐1β production by extracellular acidification through the TDAG8/cAMP pathway in mouse microglia

Interleukin‐1β (IL‐1β) is released from activated microglia and involved in the neurodegeneration of acute and chronic brain disorders, such as stroke and Alzheimer's disease, in which extracellular acidification has been shown to occur. Here, we examined the extracellular acidic pH regulation of IL‐1β production, especially focusing on TDAG8, a major proton‐sensing G‐protein‐coupled receptor, in mouse microglia. Extracellular acidification inhibited lipopolysaccharide ‐induced IL‐1β production, which was associated with the inhibition of IL‐1β cytoplasmic precursor and mRNA expression. The IL‐1β mRNA and protein responses were significantly, though not completely, attenuated in microglia derived from TDAG8‐deficient mice compared with those from wild‐type mice. The acidic pH also stimulated cellular cAMP accumulation, which was completely inhibited by TDAG8 deficiency. Forskolin and a cAMP derivative, which specifically stimulates protein kinase A (PKA), mimicked the proton actions, and PKA inhibitors reversed the acidic pH‐induced IL‐1β mRNA expression. The acidic pH‐induced inhibitory IL‐1β responses were accompanied by the inhibition of extracellular signal‐related kinase and c‐Jun N‐terminal kinase activities. The inhibitory enzyme activities in response to acidic pH were reversed by the PKA inhibitor and TDAG8 deficiency. We conclude that extracellular acidic pH inhibits lipopolysaccharide‐induced IL‐1β production, at least partly, through the TDAG8/cAMP/PKA pathway, by inhibiting extracellular signal‐related kinase and c‐Jun N‐terminal kinase activities, in mouse microglia.