Cloning of the functional promoter for human insulin-like growth factor binding protein-4 gene: endogenous regulation

The majority of the colon cancers analyzed to-date express insulin-like growth factor binding protein (IGFBP)-4, and antisense inhibition of IGFBP-4 messenger RNA (mRNA) confers a growth advantage to the cells in response to endogenous and exogenous IGFs. We recently reported a significant up-regulation of IGFBP-4 expression in a human colon cancer cell line (CaCo2) on spontaneous differentiation of the cells in culture. This suggests that the expression of IGFBP-4 may be related to growth and differentiation of colon cancer cells. To study the endogenous factors involved in the transcriptional regulation of IGFBP-4, we have isolated and sequenced the human (h) IGFBP-4 promoter. The approximately 1.3 kilobase pair (kb) 5' flanking region of the IGFBP-4 gene is GC rich and possesses several potential regulatory elements. These elements include a typical TATA box with sequence TATAA, located -299 nt from the initiation ATG codon. The cap site is located 14 nt downstream of the TATA box as determined by primer extension analysis. A 1.4-kb DNA fragment including the 1.254 kb 5' flanking region of the hIGFBP-4 gene was subcloned into a luciferase reporter vector (pGL-2 basic) either in the sense (BP-4-S-pGL) (S) or antisense (BP-4-AS-pGL) (AS) (negative control) orientation, relative to the luciferase coding sequence in the vector. CaCo2 cells were transfected with either the S or the AS vectors on days 2-10 of culture; cotransfection with the SV40-beta-Galactidose (Gal) vector was used to correct for transfection efficiency. The ratio of luciferase/beta-Gal expression by CaCo2 cells transfected with the S vectors increased significantly from days 3 and 4 to days 5 and 6 of culture, followed by a sharp decline on days 7-9, resembling the pattern of endogenous expression of IGFBP-4 by the cells; the expression of luciferase by the AS vectors remained low and insignificant. These results thus suggest that the approximately 1.4 kb 5' flanking region of the IGFBP-4 gene contains the cis elements required for regulation of the IGFBP-4 gene. Cloning and sequencing of the functional hIGFBP-4 promoter will enable us, for the first time, to study the endogenous factors/mechanisms responsible for the growth/differentiation (cell density) associated regulation of IGFBP-4 expression in colonic epithelial cells.