Increased Sp1 phosphorylation as a mechanism of hepatocyte growth factor (HGF/SF)-induced vascular endothelial growth factor (VEGF/VPF) transcription

Abstract

Hepatocyte growth factor (HGF/SF)-induced expression of vascular endothelial growth factor (VEGF/VPF) has been implicated in paracrine amplification of angiogenesis, contributing to angiogenic responses during inflammation, wound healing, collateral formation and tumor growth. We have shown previously that HGF/SF-mediated VEGF/VPF expression by keratinocytes is primarily dependent on transcriptional activation, and we mapped the HGF/SF-responsive element to a GC-rich region between bp -88 and -65. Sp1-like factors bind to this element constitutively; however the VEGF/VPF promoter is transactivated by HGF/SF in the absence of induced binding activity. In experimental approaches to clarify molecular mechanisms of Sp1-dependent VEGF/VPF gene transcription, neither HGF/SF-dependent changes in nuclear expression nor in relative DNA binding activity of Sp family members to the indicated element were observed. Thus, HGF/SF was hypothesized to induce VEGF/VPF gene transcription via increased transactivation activity of Sp1 owing to biochemical modification. In immunoprecipitation studies, HGF/SF was found to increase the amount of serine-phosphorylated Sp1, revealing a likely mechanism of HGF/SF-induced VEGF/VPF expression, as phosphorylation may enhance the transcriptional activity of Sp1. The contribution of different signaling molecules to HGF/SF-induced VEGF/VPF transcription was demonstrated by the use of chemical inhibition, of expression of kinase-deficient signaling proteins, and by the use of antisense oligonucleotides. Herein, we provide evidence that PI 3-kinase, MEK1/2 and PKC-ζ play a significant role in HGF/SF-induced VEGF/VPF promoter activation. Together, our results elucidate a critical pathway of paracrine amplification of angiogenesis, suggesting that HGF/SF-induced Sp1 phosphorylation may activate VEGF/VPF promoter activity that requires the contribution of distinct signaling molecules.

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

“…Experiments of Sp1 immunoprecipitation performed to evaluate the presence of serine-and threonine-phosphorylated proteins revealed increased intracellular amounts of serine-phosphorylated Sp1 in response to Bcl-2 overexpression. In agreement with other reports, demonstrating that Sp1 phosphorylation of serine residues correlates with enhanced transcription of "Sp1 site-dependent" genes (31,34), we hypothesized that Bcl-2 is involved in the increase of Sp1 transcriptional activity through serine phosphorylation of the transcription factor. MTR, a pharmacological inhibitor of Sp1, prevented the induction of Sp1 DNA binding activity and uPAR expression by Bcl-2, thus confirming the role of Sp1 in Bcl-2-induced uPAR expression.…”

“…Increased Sp1 phosphorylation and activity has also been proposed to result from oxidative stress-, tumor necrosis factorand hepatocyte growth factor-induced VEGF transcription (34,35,59). Thus, it is likely that this transcription factor also plays a role in Bcl-2-induced VEGF expression that we previously demonstrated (15,16).…”

“…The regulation of Sp1-dependent transcription can be affected by changes in the overall amount of Sp1 or in transactivation activity due to biochemical modifications such as phosphorylation (31)(32)(33)(34)(35)(36). To address this issue, we analyzed Sp1 protein expression, stability, and phosphorylation in M14 parental cells, the MN8 control clone, and the MB5 and MB6 bcl-2 transfectants grown in normoxia or exposed to hypoxia for 24 h.…”

Interleukin‐6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1

“…46 IL-6 is known to signal primarily via the STAT and MAPK pathways. 35 In contrast to HGF-induced VEGF transcription, where Sp1 phosphorylation is dependent on PI3-K and MEK1/2 signalling, 47 pretreatment of NIH3T3 cells with either PD98059 or LY294002 led only to a 30% reduction of IL-6-triggered VEGF transcription (data not shown), suggesting that the MAPK and PI3-K/Akt signaling pathways are only partially involved. However, transfection of NIH3T3 cells and U87MG cells with dominant-negative STAT3 reduced IL-6-mediated VEGF expression to control levels, attesting that STAT3 is absolutely required for IL-6-regulated VEGF expression.…”

The Tetraspanin CD81 Protein Increases Melanoma Cell Motility by Up-regulating Metalloproteinase MT1-MMP Expression through the Pro-oncogenic Akt-dependent Sp1 Activation Signaling Pathways

“…Because threonine 453 phosphorylation of Sp1 was shown to increase its DNA binding and/or transcriptional activities toward target genes including VEGF (37-39), increased threonine phosphorylation of Sp1 by CD81 signaling is likely a mechanism of CD81-induced MT1-MMP transcription in melanoma cells. Meanwhile, the ERK, Akt, and JNK pathways are implicated in increasing Sp1 phosphorylation in various cell systems (38,40). Interestingly, Carloni et al (41) found that CD81 increases synthesis of phosphoinositides by activating the associated PI4K, leading to phosphoinositide-facilitated membrane translocation of Shc in hepatic tumor cells.…”