bcl-2 Induction of Urokinase Plasminogen Activator Receptor Expression in Human Cancer Cells through Sp1 Activation

Abstract

We have previously demonstrated that Bcl-2 overexpression in human breast carcinoma and melanoma cells synergizes with hypoxia to increase angiogenesis through up-regulation of vascular endothelial growth factor. In this work we demonstrated, for the first time, that Bcl-2 overexpression in cancer cells exposed to hypoxia modulates urokinase plasminogen activator receptor (uPAR) expression through Sp1 transcription factor and that the extracellular signal-regulated kinase (ERK) pathway plays a role in Sp1 transcriptional activity. In particular, an increase in uPAR protein and mRNA expression was found in melanoma bcl-2 transfectants grown under hypoxia when compared with control cells, and a decrease of uPAR protein expression was induced by treatment of cells with specific bcl-2 antisense oligonucleotides. Up-regulation of uPAR expression was accompanied by increased Sp1 protein expression, stability, serine phosphorylation, and DNA binding activity. Treatment of cells with mitramycin A, an inhibitor of Sp1 activity, confirmed the role of Sp1 transcriptional activity in uPAR induction by Bcl-2. The contribution of the ERK pathway in Sp1-increased transcriptional activity was demonstrated by the use of chemical inhibition. In fact, ERK kinase activation was induced in Bcl-2-overexpressing cells exposed to hypoxia, and the ERK kinase inhibitor UO126 was able to down-regulate Sp1 phosphorylation and DNA binding activity. Using a human breast carcinoma line, we obtained data supporting our findings with melanoma cells and identified a link between the induction of Sp1 and uPAR expression as a common bcl-2-controlled phenomenon in human tumors. In conclusion, our results strongly indicate that up-regulation of uPAR expression by Bcl-2 in hypoxia is modulated by Sp1 DNA binding activity through the ERK signaling pathway.Angiogenesis is a fundamental process required for tumor growth, invasion, and metastasis and is strongly induced by hypoxia. In fact, several cell types including tumor cells, macrophages, and endothelial cells respond to hypoxia by producing angiogenic factors, fibrinolytic factors, and adhesion molecules involved in pathologic angiogenesis (1-5). Four sequential steps can be distinguished during angiogenesis: the degradation of the basement membrane and interstitial matrix, endothelial cell migration, endothelial cell proliferation, and the formation of tubular structures with a lumen and a new basement membrane (6). Three of these steps critically depend on proteolytic activity generated by the matrix metalloproteinases and the plasminogen activator/plasmin system. In particular, the role of urokinase plasminogen activator receptor (uPAR) 1 in tumor cell invasion and migration and in the formation of new microvascular structures has been largely demonstrated (7-9).Angiogenesis is also controlled by alterations in oncogene and tumor suppressor gene expression (10 -14). In this context, we previously demonstrated that the bcl-2 oncogene increases in vitro and in vivo angiogenesis in two different t...

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function

“…Our previous data demonstrate that Bcl-2 overexpression increases the expression of NF-kB and ERK 1/2, two molecules involved in the regulation of hTERT. 35,36 The Bcl-2 inhibitor HA14-1, although unable to inhibit the overall telomerase activity, altered intracellular hTERT protein localization, indicating that modulation of Bcl-2 function interferes with telomerase function by inducing nuclear export of hTERT. Recently, translocation of hTERT has been described as an additional mechanism for post-transcriptional regulation.…”

Overexpression of Sp1 leads to p53‐dependent apoptosis in cancer cells

“…7-12 Although Sp1 regulates a number of oncogenes such as those coding for the vascular endothelial growth factor, urokinase plasminogen activator (uPA), uPA receptor, and epithelial growth factor receptor, [13][14][15] it also induces the expression of many tumor suppressor genes such as p27 kip1 , p21 WAF1/CIP1 , p16 INK4a , and pp2a-c. [16][17][18] In addition, the promoters of many proapoptotic genes have been found to contain Sp1-binding elements; for instance, fas, fas ligand, bax, and caspase 3.…”

“…Sp1 commonly undergoes posttranslational modifications such as phosphorylation, glycosylation, acetylation, ubiquitination, and sumoylation. [2][3][4][5][6] Numerous studies have documented that Sp1 activity and/or expression levels are elevated in human pancreatic cancer, breast cancer, colorectal cancer, gastric carcinoma, hepatocellular carcinoma, and thyroid carcinoma.7-12 Although Sp1 regulates a number of oncogenes such as those coding for the vascular endothelial growth factor, urokinase plasminogen activator (uPA), uPA receptor, and epithelial growth factor receptor, [13][14][15] it also induces the expression of many tumor suppressor genes such as p27 kip1 , p21 WAF1/CIP1 , p16 INK4a , and pp2a-c. [16][17][18] In addition, the promoters of many proapoptotic genes have been found to contain Sp1-binding elements; for instance, fas, fas ligand, bax, and caspase 3.19,20 Interestingly, it has been found that Sp1 can also induce the expression of many antiapoptotic genes (such as survivin, bcl-x, and bcl-w).20,21 Moreover, several recent studies have indicated that the overexpression of Sp1 causes apoptotic cell death in nontransforming cells, but the downregulation of the overexpressed Sp1 protein in human fibrosarcoma cell lines inhibits tumor formation. 13,22 Therefore, the effect of Sp1 overexpression on apoptosis regulation in transforming cells remains controversial, and the detailed mechanism of this process remains unclear.…”

Activated platelets increase fibrinolysis of mesenchymal progenitor cells

“…29,30 The signaling pathway involved in the expression of plasminogen activators and the respective inhibitors has been extensively studied. [31][32][33][34] However, the possible role of these pathways to mediate the effects of activated platelets on the plasminogen activation system by mesenchymal progenitors remains unknown. Most of the effects on the plasminogen activator system seem to involve mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 as well as phosphatidylinositol 3-kinase (PI3K)/AKT pathways in other cells.…”

“…Most of the effects on the plasminogen activator system seem to involve mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 as well as phosphatidylinositol 3-kinase (PI3K)/AKT pathways in other cells. [31][32][33][34] Moreover, activated platelets were reported to enhance MAPK signaling in BMSC, suggesting a possible association with the plasminogen activation system of mesenchymal progenitors. progenitor cells.…”