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. 2003 Jan;23(2):543-54.
doi: 10.1128/MCB.23.2.543-554.2003.

The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway

Jong-In Park  1 , Christopher J Strock, Douglas W Ball, Barry D Nelkin
Affiliations

The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway

Jong-In Park et al. Mol Cell Biol. 2003 Jan.

Abstract

Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.

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Figures

FIG. 1.
FIG. 1.
The effects of conditioned media on MTC cells. TT cells were treated with the conditioned medium Raf-E2-CM and the control conditioned media pLNCX-EtOH-CM, pLNCX-E2-CM, and Raf-EtOH-CM. Cells were observed for changes in morphology (A), growth (B), incorporation of [3H]thymidine into DNA in 6 h (C), and expression of phosphorylated ERK1/2 and RET (Western blotting) or calcitonin genes (Northern hybridization) after 2 days (D). Data (mean ± standard error) are from a representative experiment performed in triplicate. P value is <0.001 for Raf-E2-CM compared to Raf-EtOH-CM on a cell growth curve (one-way analysis of variance). Experiments were repeated at least three times with similar results.
FIG.2.
FIG.2.
LIF is the autocrine growth inhibitor induced by Raf. (A) Conditioned medium was concentrated by ultrafiltration, dialyzed (sample), and subjected to a series of protein purification columns, consisting of the anion exchanger unoQ6 coupled with heparin-Sepharose (Q/Heparin), octyl-Sepharose coupled with butyl-Sepharose (Octyl/Butyl; flow through), the cation exchanger unoS1 (S), and Superdex G200 gel filtration (GF). Active fractions from each column were resolved by SDS-PAGE and visualized by silver staining. (B) Expression of LIF upon Raf activation was investigated by RT-PCR of total RNA from TTpLNCX or TTRaf cells treated with ethanol or estradiol (top and middle panels) and by Western blot detection of LIF in cell culture media (bottom panel). (C to E) TT cells were treated with the control conditioned media, Raf-E2-CM, LIF-neutralized Raf-E2-CM (Raf-E2-CM+anti-LIF), and LIF. Cells were also pretreated with anti-gp130 blocking antibody prior to Raf-E2-CM treatment (Raf-E2-CM+anti-gp130). Cells were then observed for changes in morphology (C), growth (D), and expression of phosphorylated ERK1/2, RET (Western hybridization), or calcitonin genes (Northern hybridization) after 2 days (E). Data (mean ± standard error) are from a representative experiment performed in triplicate. P value is <0.001 for Raf-E2-CM+anti-LIF and Raf-E2-CM+anti-gp130 compared to Raf-E2-CM on cell growth curve (one-way analysis of variance). Experiments were repeated at least three times with similar results.
FIG. 3.
FIG. 3.
STAT3 is essential for LIF action. (A) TT cells were treated with conditioned media or LIF for 2 days and examined by Western blot hybridization for STAT3 expression. (B to D) TTSTAT3-DN cells or the control TTpcDNA3.1 cells were treated for 2 days with LIF and observed for changes in morphology (B), growth (C), and downregulation of RET expression (D). The presence of STAT3-DN was detected by Western analysis of the C-terminal FLAG tag. Data (mean ± standard error) are from a representative experiment performed in triplicate. P value is <0.0001 for STAT3DN-LIF compared to pcDNA3.1-LIF on a cell growth curve (one-way analysis of variance). Experiments were repeated at least three times with similar results. Experiments were also done with other independent stably transfected clones with similar results (data not shown).
FIG. 4.
FIG. 4.
MEK1/2 is essential for production of LIF but is not required for LIF action. (A and B) TT cells were treated for 2 days with LIF in the presence of the MEK1/2 inhibitor U0126 (10 μM) and observed for changes in expression of phosphorylated ERK1/2, RET, and phosphorylated STAT3 by Western blotting (A) and morphology (B). (C) TTRaf cells were treated for 2 days with estradiol in the presence of U0126 and observed for changes in expression of phosphorylated ERK1/2 and LIF by Western blotting. (D and E) TT cells were infected with adenoviruses containing constitutively active Ras V12 or Raf BXB in the presence of U0126. After 2 days, cells were observed for LIF expression by RT-PCR (D) and STAT3 activation (E). The data presented are fold increases of activity upon treatment. P values are <0.005 for Ras, Raf, LIF, and LIF+U0126 compared to the control (one-way analysis of variance). Experiments were repeated at least three times with similar results. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIG. 5.
FIG. 5.
Raf has an intracellular growth-inhibitory pathway independent of LIF/JAK/STAT3. TTRaf cells were treated for 1 day with estradiol in the presence of the anti-LIF neutralizing antibody or anti-gp130 blocking antibody and observed for changes in expression of RET and phosphorylation of STAT3 by Western blot hybridization (A) and morphology (B). (C) TT or TTRaf cells infected for 2 days with AdSTAT3-DN were treated with LIF or estradiol, respectively, and observed for changes in morphology and growth. Cells were also infected with an equal dose of AdGFP control virus for comparison. Similar infection ratio was checked by green fluorescent protein expression. Similarity in levels of STAT3-DN expression in TT and TTRaf cells was confirmed by Western analysis of the C-terminal FLAG tag (data not shown). Experiments were repeated at least three times with similar results.
FIG. 6.
FIG. 6.
LIF/JAK/STAT3 signaling in small cell lung cancer cell lines. (A) Production and secretion of LIF was analyzed by Western hybridization of cell culture media from H209 and H209Raf cells or DMS53 and DMS53Raf cells treated with estradiol for 2 days. (B) H209 or DMS53 cells were treated for 2 days with LIF or Raf-E2-CM produced from TTRaf cells, and phosphorylation of tyrosine 705 residue of STAT3 was analyzed. (C) H209 or DMS53 cells were treated for 2 days with LIF, and activation of STAT3 was analyzed. The data presented are fold increases of activity upon LIF treatment. Data (means ± standard deviations) are from a representative experiment performed in triplicate. Experiments were repeated at least three times with similar results.
FIG. 7.
FIG. 7.
Signal transduction pathways for Ras/Raf-mediated growth inhibition and differentiation. The potential growth-inhibitory signaling by Ras/Raf is depicted. The autocrine-paracrine signaling diverges from the intracellular signaling distal to MEK/ERK. The extracellular pathway mediates its effect through LIF expression and activation of JAK-STAT3.
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