Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts.

Abstract

Insulin-like growth factor binding protein 3 (IGFBP-3) mRNA levels were consistently higher in both senescent normal human diploid fibroblasts (HDFs) at late passage (old cells) and prematurely senescent IDFs from a subject with Werner syndrome (WS) during serum depletion and repletion of growth medium and during prollferation from sparse to high-density inhibited cultures, compared to normal early-passage (young) HDFs. However, IGFBP-3 protein accumulated to higher levels in conditioned medium of old cells than in medium of WS and young cells, in that order, under the same conditions. Insulin-like growth factor I (IGF-I) was not detected in naive medium or in any of the media conditioned by these three cOll types, whereas IGF-ll was detectable in serumrepleted medium and remained relatively constant. Thus, molar ratios of IGFBP-3/IGF-II were constently higher in old and WS cells and increased substantially as all three cell types became quiescent, due to either serum depletion or hig cell density. These data are consistent with either an adaptive or a causal role for IGFBP-3 protein in the senescent and quiescent growth arrest of HDFs.Human diploid fibroblasts (HDFs) have a finite replicative life-span (1), and this system is now widely used as a model for the study of biological aging (2, 3). Thus, the number of cumulative mean population doublings (MPD) prior to senescent arrest of HDFs is inversely proportional to the age of the donor. Moreover, persons with various inherited disorders of premature aging, such as Werner syndrome (WS), give rise to HDFs with an abbreviated replicative life-span (4, 5). The mechanism for loss. of HDF replicative ability is unknown but positive and negative growth regulatory factors appear to be involved (6).A prominent characteristic of senescent HDFs, whether derived from normal donors or from subjects with WS, is unresponsiveness to the action of various polypeptide mitogens present in fetal bovine serum (FBS) including insulinlike growth factor I (IGF-I), platelet-derived growth factor, and epidermal growth factor (7-12). However, no significant alterations have been detected in the number of binding sites or in the binding affinity of IGF-I and these other factors after correction for the increased surface area of enlarged senescent HDFs (ref. 11; see also ref. 3). IGF-I unresponsiveness, therefore, would appear to reside in either a postreceptor mechanism and/or in a prereceptor block that restricts growth factor bioavailability.The presence in vertebrate sera of binding proteins for the IGF family (IGFBPs) has recently generated considerable interest (13). Six structurally distinct IGFBPs have now been recognized (14). In adult human serum, the major species is IGFBP-3, which exists as part of a ternary, growth hormonedependent complex of %150 kDa. In brief, the components of this complex are an acid-stable glycoprotein (IGFBP-3), which can exist in at least two glycosylated forms of 40-50 kDa; IGF-I or IGF-II, which bind to IGFBP-3 with equally high affinity; a...

Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras

“…In senescent human ®broblasts, the levels of p21 are increased (Noda et al, 1994;Tahara et al, 1995;Alcorta et al, 1996), together with those of other p53 transcriptional targets, such as IGFBP-3 (Goldstein et al, 1991;Buckbinder et al, 1995). However, it is not well established which is the relative contribution of p53 in the upregulation of p21 during senescence (Bond et al, 1995;Tahara et al, 1995;Medclaf et al, 1996;Gallimore et al, 1997).…”

In contrast to lung fibroblasts – no signs of senescence in skin fibroblasts of patients with emphysema

“…In addition, we found an increased staining for senescence-associated beta-galactosidase (β-gal, (Dimri et al 1995)), as well as increased mRNA expression of the insulin-like growth factor-binding proteins (IGFBP)-3 and -rP1 in these cells (Müller et al 2006). Both proteins have been shown earlier to be senescenceassociated (Goldstein et al 1991;Swisshelm et al 1995;Wilson et al 2002).…”

Characterization of a Novel Positive Transcription Regulatory Element That Differentially Regulates the Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Gene in Senescent Cells

“…Increased expression of IGFBP-3 is also seen upon serum withdrawal or contact inhibition-induced cell cycle arrest (25). To test whether the increased binding activity was specific to senescence, we incubated the 30-bp element with extracts from young proliferating cells, young quiescent cells, and subconfluent senescent cells.…”

“…Compared with young cells, senescent fibroblasts show substantially up-regulated IGFBP-3 mRNA and protein (24 -26), and the protein also accumulates in the growth medium of young cells to levels directly correlated with the chronological age of the donor (25)(26)(27). Furthermore, Grigoriev et al (28) found that IGFBP-3 accumulation in the medium of senescent human diploid fibroblasts can bind and sequester IGFs and thereby attenuate the response of senescent cells to IGF-I.…”