Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells

Abstract

In this project, we studied the gene regulation of 15-lipoxygenase 2 (15-LOX2), the most abundant arachidonate-metabolizing LOX in adult human prostate and a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells. Through detailed in silico promoter examination and promoter deletion and activity analysis, we found that several Sp1 sites (i.e., three GC boxes and one CACCC box) in the proximal promoter region play a critical role in regulating 15-LOX2 expression in NHP cells. Several pieces of evidence further suggest that the Sp1 and Sp3 proteins play a physiologically important role in positively and negatively regulating the 15-LOX2 gene expression, respectively. First, mutations in the GC boxes affected the 15-LOX2 promoter activity. Second, both Sp1 and Sp3 proteins were detected in the protein complexes that bound the GC boxes revealed by electrophoretic mobility shift assay. Third, importantly, inhibition of Sp1 activity or overexpression of Sp3 both inhibited the endogenous 15-LOX2 mRNA expression. Since 15-LOX2 is normally expressed in the prostate luminal epithelial cells, we subsequently explored whether androgen/androgen receptor may directly regulate its gene expression. The results indicate that androgen does not directly regulate 15-LOX2 gene expression. Together, these observations provide insight on how 15-LOX2 gene expression may be regulated in NHP cells.

Cell-autonomous induction of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) contributes to replicative senescence of human prostate progenitor cells

“…One possibility is Figure 6 Enforced expression of 15-LOX2 or 15-LOX2sv-b in early-passage NHP7 cells induces cell-cycle arrest and a senescence-like phenotype. NHP7 cells at P2 were plated at clonal density were either untransduced or infected with pBabe-EGFP, pBabe15LOX2-EGFP (two clones used, that is, FL-3 and FL-4; see Table 2), or pBabe15LOXsv-b-EGFP (two clones used, that is, SVb-1 and SVb-2; see Table 2 15-LOX2 and normal human prostate epithelial cell senescence B Bhatia et al through increased Sp1 transcriptional activity (Tang et al, 2004). In support, 15-LOX2 promoter constructs with the Sp1 sites mutated, when transfected into latepassage NHP6 cells, possess much lower promoter activities compared to the constructs with the intact Sp1 sites (Tang et al, 2004).…”

“…To determine whether 15-LOX2 induction resulted from transcriptional activation, we first measured the 15-LOX2 promoter activity in NHP6 cells transfected with the p15LOX2 (À726/ þ 80)-luc reporter construct (Tang et al, 2004). As shown in Figure 4B, increasing 15-LOX2 promoter activity was observed in NHP6 cells with increasing passages.…”

“…Luciferase reporter plasmids and anti-15-LOX2 antibody were previously described (Bhatia et al, 2003;Tang et al, 2004). Other antibodies used in this study include: polyclonal anti-CK5 (BabCO), monoclonal anti-CK18 (clone RGE53; BD PharMingen), monoclonal anti-p63 (clone 4A4), PSA and AR (Santa Cruz), 2 monoclonal anti-CD44 (clone G44-26 from BD PharMingen and DF1485 from Santa Cruz), polyclonal anti-hTERT (AbCAM), monoclonal anti-CD57 (clone NK-1) and a2b1 (PharMingen), and monoclonal anti-PAP (clone PASE/4LJ; Dako).…”

“…NHP2, NHP4, and NHP6 cells (Tang et al, 2002(Tang et al, , 2004) and NHP7 cells (Clonetics) were cultured on collagen-coated dishes in serum-and androgen-free, PrEBM medium supplemented with insulin, EGF, hydrocortisone, bovine pituitary extract, and cholera toxin, and used at passage 2-8. Luciferase reporter plasmids and anti-15-LOX2 antibody were previously described (Bhatia et al, 2003;Tang et al, 2004).…”

Downregulation of 15-lipoxygenase 2 by glucocorticoid receptor in prostate cancer cells

“…Recently there were two other studies focused on the promoter regulation of 15-LOX-2 gene. Tang et al (9) reported that Sp1 positively and Sp3 negatively regulate and androgen does not regulate 15-LOX-2 gene expression in normal human prostate epithelial cells. Subbarayan et al (5) reported that 15-LOX-2 can be negatively regulated by its product 15-(S)-hydroxyeicosatetraenoic acid and PPARÁ through a PPAR half-site present in the 15-LOX-2 promoter region (-560 to -596).…”