TGF‐β₁ inhibits DNA synthesis and phosphorylation of the retinoblastoma gene product in a rat liver epithelial cell line

Abstract

In the rat liver epithelial cell line, WB, the ability of TGF-beta 1 to inhibit DNA synthesis was shown to correlate with its ability to inhibit phosphorylation of the protein product of the retinoblastoma susceptibility gene, pRb. When WB cells were serum-starved, then refed with serum-containing medium, a peak of DNA synthesis occurred at about 18 h. Autoradiographs showed that 43.6% of cell nuclei could be labeled with 3H-thymidine at this time. When TGF-beta 1 was added simultaneously with serum, it blocked DNA synthesis and reduced the number of labeled nucleii to 6.3%. Cells treated with serum alone for 18 h also showed a pronounced increase in the highly phosphorylated form of pRb, as shown by mobility shifts in immunoblots, and in active phosphorylation of pRb, as shown by 32P incorporation. Simultaneous addition of TGF-beta 1 with serum abolished both 32P incorporation into pRb and its mobility shift on immunoblots. The effect of TGF-beta 1 on DNA synthesis measured at 18 h was sharply reduced if the cells were incubated with serum for 8 h (and thus allowed to enter S) before the addition of TGF-beta 1. If TGF-beta 1 was added after 8 h of serum treatment, its ability to inhibit pRb phosphorylation at 18 h was unchanged. If TGF-beta 1 was added after 13 h of serum treatment, its effects on pRb phosphorylation were reduced. Thus, as the cell population moved into S, the ability of TGF-beta 1 to inhibit both pRb phosphorylation and DNA synthesis was lost. In higher passages of WB cells the dose-response for inhibition of DNA synthesis by TGF-beta 1 was shifted to the right. Inhibition of pRb phosphorylation by TGF-beta 1 was also lost in higher passage WB cells. Thus, the passage-dependent loss of sensitivity to inhibition of DNA synthesis accompanied the loss of sensitivity to inhibition of pRb phosphorylation. Since the phosphorylation of pRb is believed to be required for the progression of cells from G1 to S, inhibition of pRb phosphorylation may be either a cause or a consequence of the G1 arrest of WB cells by TGF-beta 1.

Growth inhibition of human keratinocytes by MC903 (calcipotriol) is linked to dephosphorylation of retinoblastoma gene product

“…TG¥-fi\ is known to inhibit pho.sphorylation of pRB in rat liver epithelial cell line [16], mink lung epithelial cell [22] and human keratinocytes [23]. These results may support the hypothesis that the effect of MC903 and 1,25(OH)2D3 is closely related to pRB.…”

“…Its gene was first discovered to be a gene in which mutational inactivation resulted in the development of ocular tumors in children [10]. The pRB is dephosphorylated in Gl phase and becomes multiplephosphorylated near the Gl/S boundary; it remains highly phosphorylated through G2 and early M. Then pRB is dephosphorylated as the cell moves into late M or early Gl phase [13][14][15][16]. In addition, pRB is involved in the growth regulation of normal eells, namely cell eyele regulation.…”

The concomitant apoptosis and EMT underlie the fundamental functions of TGF-β

“…TGF-β can function as a tumor promoting factor by increasing cell metastatic motility, survival ability, and angiogenesis. About two decades ago, the most widely known cellular effect of TGF-β was the induction of cell cycle arrest that led to the inhibition of the proliferation of many normal epithelial cell types [16][17][18]. Similarly, the apoptosis induction effect of TGF-β in normal epithelial cells has also been identified during that period [19][20][21][22].…”

Negative control of cell proliferation in eukaryotes

“…As mentioned previously, inhibition of epithelial cell proliferation by TK3F-B at the GI/S transition is accompanied by dephosphorylation of the retinoblastoma gene product (pRB) (Laiho et al 1990, Whitson & Itakura 1992 which implies at least some role for phosphatases. Hyperphosphorylated pRB binds less tightly to nuclei than does the growth-inhibitory hypophosphorylated form (Templeton 1992) and is more easily extracted (Mittnacht & Weinberg 1991).…”

“…Inhibition, by TGF-/3, of epithelial cell proliferation at the G,/S transition is accompanied by dephosphorylation of the retinoblastoma gene product (pRB) (Laiho et al 1990, Whitson & Itakura 1992) and a decrease in the histone H1 kinase activity of ~3 4 '~'~ (Howe, Draetta & Leof 1991). Northern blot analysis of human keratinocytes (Geng & Weinberg 1993) revealed that EF-B could suppress cell cycle progression in two distinct ways.…”