The regulation of cyclin D1 expression in senescent human fibroblasts

Abstract

To clarify the molecular mechanisms of cyclin D1 expression during in vitro cellular aging, we investigated the binding of nuclear protein factors to the cyclin D1 gene promoter domain in young and senescent normal human fibroblasts. The cyclin D1 promoter binding activities of nuclear protein factors from young and senescent cells were examined by the gel mobility shift assay. Our findings revealed that (i) the binding of a specific nuclear factor to the enhancer element was very weak in senescent cells; (ii) the binding of a specific nuclear factor to the CRE, which is independent of cell growth, was unchanged between young and senescent cells; (iii) nuclear factors from senescent cells did not bind to the presumptive silencer element; (iv) the binding of specific factors to the Inr (transcription initiation region) and E2F increased with growth stimulation in young cells and was weakly detectable in senescent cells; and (v) the binding of Sp1 to its promoter element occurred only in senescent cells. The analysis of the silencer element by the gel mobility shift assay revealed that the essential sequence required for binding of specific factors to the silencer element was TTTAAT. The molecular weight of the binding factor to the silencer element was determined to be approximately 35 kDa by the Southwestern blotting and UV cross-linking assay. Thus, we postulated that the observed increase of cyclin D1 expression during cellular aging is due to an increase in the binding activity of specific nuclear protein factors to an enhancer element, Sp1, and a decrease in binding to a silencer element in senescent cells.