Dihydrosphingosine 1-phosphate stimulates MMP1 gene expression via activation of ERK1/2-Ets1 pathway in human fibroblasts.

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes formation of important regulators of inter- and intracellular signaling, sphingosine-1 phosphate (S1P), and dihydrosphingosine 1-phosphate (dhS1P). In this study, we investigated the role of SphK1 in the regulation of expression of matrix metalloproteinase 1 (MMP1) in dermal fibroblasts, a key event in regulation of extra cellular matrix. We show that overexpression of SphK1 up-regulated MMP1 protein, MMP1 mRNA, and MMP1 promoter activity, and this action of SphK1 required activation of the ERK1/2-Ets1 and NF-kappaB pathways. Furthermore, experiments using SphK1 specific siRNA demonstrated that SphK1 is required for the TNF-alpha stimulation of MMP1. Additional data revealed a specific role of dhS1P, and not S1P, as a mediator of SphK1-dependent activation of ERK1/2 and up-regulation of MMP1. The stimulatory effect of dhS1P was sensitive to pertussis toxin, suggesting a possible involvement of a G-protein-coupled receptor. In contrast, S1P, but not dhS1P, stimulated the induction of COX-2, which demonstrated selective actions of these two closely related bioactive lipids. In conclusion, this study describes a novel mode of SphK1 signaling through generation of dhS1P with a key role in mediating transcriptional responses to TNF-alpha. This is the first report of selective function of dhS1P as compared with the better studied S1P.

Caspase-1 is a direct target gene of ETS1 and plays a role in ETS1-induced apoptosis.

ETS1, the founding member of Ets transcriptional factor family, plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. Previous work has shown that ETS1 represses tumorigenicity of colon carcinoma cells in vivo, and that the p42-ETS1 protein bypasses a defect in apoptosis in colon carcinoma cells through the up-regulation of caspase-1 expression. In this report, we show that expression of p42-ETS1 inhibits tumorigenicity of colon cancer DLD-1 cells through induction of apoptosis in vivo. In support of the hypothesis that caspase-1 might be a target involved in the sensitization of DLD-1 cells to Fas-induced apoptosis by ETS1, overexpression of caspase-1 bypasses Fas-induced apoptosis in these cells as well. Furthermore, ETS1-mediated apoptosis was observed in MOP8 cells, a transformed mouse NIH3T3 cell line. To determine whether ETS1 activates the transcription of caspase-1, luciferase reporters driven by the wild-type and mutant caspase-1 promoters were generated. Both p51-ETS1 and p42-ETS1 transactivated the caspase-1 transcription and a functional Ets binding site is identified in the caspase-1 promoter. Wild-type caspase-1 promoter (pGL3-ICE) was strongly transactivated by ETS1 and this transactivation was dramatically diminished by the mutation of the potential Ets binding site (-525 bp). In addition, electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed complex formation between this binding site and ETS1 proteins. Taken together, ETS1 transcriptionally induces the expression of caspase-1; as such, the regulatory control of caspase-1 expression by ETS1 may underlie the apoptotic susceptibility modulated by ETS1 in specific tumor cells.

SP100 inhibits ETS1 activity in primary endothelial cells.

SP100 was first identified as a nuclear autoimmune antigen and is a constituent of the nuclear body. SP100 interacts with the ETS1 transcription factor, and we have previously shown that SP100 reduces ETS1-DNA binding and inhibits ETS1 transcriptional activity on the MMP1 and uPA promoters. We now demonstrate that SP100 expression is upregulated by interferons, which have been shown to be antiangiogenic, in primary endothelial cells. As ETS1 is functionally important in promoting angiogenesis, we tested the hypothesis that ETS1 activity is negatively modulated by SP100 in endothelial cells. SP100 directly antagonizes ETS1-mediated morphological changes in human umbilical vein endothelial cell (HUVEC) network formation and reduces HUVEC migration and invasion. To further understand the functional relationship between ETS1 and SP100, cDNA microarray analysis was utilized to assess reprogramming of gene expression by ETS1 and SP100. A subset of the differentially regulated genes, including heat-shock proteins (HSPs) H11, HSPA1L, HSPA6, HSPA8, HSPE1 and AXIN1, BRCA1, CD14, CTGF (connective tissue growth factor), GABRE (gamma-aminobutyric acid A receptor epsilon), ICAM1, SNAI1, SRD5A1 (steroid-5-alpha-reductase 1) and THY1, were validated by real-time PCR and a majority showed reciprocal expression in response to ETS1 and SP100. Interestingly, genes that are negatively regulated by ETS1 and upregulated by SP100 have antimigratory or antiangiogenic properties. Collectively, these data indicate that SP100 negatively modulates ETS1-dependent downstream biological processes.

SP100 expression modulates ETS1 transcriptional activity and inhibits cell invasion.

The ETS1 transcription factor is a member of the Ets family of conserved sequence-specific DNA-binding proteins. ETS1 has been shown to play important roles in various cellular processes such as proliferation, differentiation, lymphoid development, motility, invasion and angiogenesis. These diverse roles of ETS1 are likely to be dependent on specific protein interactions. To identify proteins that interact with ETS1, a yeast two-hybrid screen was conducted. Here, we describe the functional interaction between SP100 and ETS1. SP100 protein interacts with ETS1 both in vitro and in vivo. SP100 is localized to nuclear bodies and ETS1 expression alters the nuclear body morphology in living cells. SP100 negatively modulates ETS1 transcriptional activation of the MMP1 and uPA promoters in a dose-dependent manner, decreases the expression of these endogenous genes, and reduces ETS1 DNA binding. Expression of SP100 inhibits the invasion of breast cancer cells and is induced by Interferon-alpha, which has been shown to inhibit the invasion of cancer cells. These data demonstrate that SP100 modulates ETS1-dependent biological processes.

EAPII interacts with ETS1 and modulates its transcriptional function.

Ets proteins constitute a family of conserved sequence-specific DNA-binding proteins and function as transcription factors. ETS1 plays important roles in differentiation, lymphoid cell development, invasiveness and angiogenesis. Such diverse roles of ETS1 are likely to be dependent on its associated proteins. A yeast two-hybrid screen was conducted and here we describe a novel ETS1 interacting protein designated as ETS1-associated protein II (EAPII). EAPII protein interacts with ETS1 and other Ets proteins (ETS2 and FLI1) both in vitro and in vivo. Indirect immunofluorescence demonstrated that EAPII is predominately localized to the nucleus of mammalian cells. EAPII negatively modulates ETS1 transcriptional activity and attenuates synergistic transactivation by ETS1 and AP-1. Significantly, re-expression of EAPII inhibits the migration of epithelial cancer cells, but does not affect cell viability. Therefore, EAPII is a novel ETS1 modulator that regulates specific aspects of the ETS1 functions.