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RAE-1 ligands for the NKG2D receptor are regulated by E2F transcription factors, which control cell cycle entry.

Jung H, Hsiung B, Pestal K, Procyk E, Raulet DH - J. Exp. Med. (2012)

Bottom Line: Raet1 genes are directly transcriptionally activated by E2F family transcription factors, which play a central role in regulating cell cycle entry.Induction of RAE-1 occurred in primary cell cultures, embryonic brain cells in vivo, and cells in healing skin wounds and, accordingly, wound healing was delayed in mice lacking NKG2D.Transcriptional activation by E2Fs is likely coordinated with posttranscriptional regulation by other stress responses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cell Biology, 489 Life Sciences Addition, University of California, Berkeley, Berkeley, CA 94720, USA.

ABSTRACT
The NKG2D stimulatory receptor expressed by natural killer cells and T cell subsets recognizes cell surface ligands that are induced on transformed and infected cells and facilitate immune rejection of tumor cells. We demonstrate that expression of retinoic acid early inducible gene 1 (RAE-1) family NKG2D ligands in cancer cell lines and proliferating normal cells is coupled directly to cell cycle regulation. Raet1 genes are directly transcriptionally activated by E2F family transcription factors, which play a central role in regulating cell cycle entry. Induction of RAE-1 occurred in primary cell cultures, embryonic brain cells in vivo, and cells in healing skin wounds and, accordingly, wound healing was delayed in mice lacking NKG2D. Transcriptional activation by E2Fs is likely coordinated with posttranscriptional regulation by other stress responses. These findings suggest that cellular proliferation, as occurs in cancer cells but also other pathological conditions, is a key signal tied to immune reactions mediated by NKG2D-bearing lymphocytes.

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Proliferation is required for RAE-1ε expression on primary fibroblasts. (A) Freshly prepared B6 adult mouse tail fibroblasts were cultured in media supplemented with 2 or 10% serum for ∼3–7 d and harvested for flow cytometry analysis. (B and C) Fibroblasts that had been cultured for >1 mo were serum starved for 1–3 d and processed for staining (B) or RNA analysis (C). Means ± SD are shown. ***, P < 0.0005, unpaired Student’s t test. (D) Samples of serum-starved cells were supplemented with 10% serum and cultured for an additional 3 d. Data in A–D are representative of at least four experiments. (E) Proliferating (Untreated) fibroblasts, or fibroblasts subjected to serum starvation or treatment with LY294002, Roscovitine, or DMSO for 3 d, were stained with 7-AAD and Annexin-V to detect dead and apoptotic cells. (F) Fibroblasts that were serum starved for 3 d were fed with serum for 2 or 3 d in the presence of BrdU to label proliferating cells, before staining the cells intracellularly with BrdU antibodies. (G) Proliferating fibroblasts were serum starved for 3 d before adding 10% serum and BrdU (bottom) or BrdU only (top). After 24 h, cells were stained with BrdU and RAE-1ε antibodies. Gated BrdU+ and BrdU− cells were examined for RAE-1ε expression after excluding 7-AAD+ (dead) cells. (H) Proliferating fibroblasts were stained with RAE-1 antibody, permeabilized, and stained with propidium iodide. Cells in G1, S, or G2 phases of the cell cycle (left) were examined for RAE-1ε expression (right). (I) Freshly prepared tail cells were cultured on fibronectin-coated plates in DMEM supplemented with 10% serum, 100 ng/ml EGF, or no growth factor (−EGF) for 3 d. Cell counts (right, cells/culture) and RAE-1ε staining (left) are shown. Data in E–I are representative of two experiments. (J) Proliferating fibroblasts (Prol) or fibroblasts that were serum starved for 3 d (Starved) were used as target cells for IL-2–activated natural killer cells (LAKs) prepared from NKG2D-deficient (KO) mice or NKG2D WT littermate controls. NKG2D antibody was added to a reaction of WT LAKs with proliferating target cells to block the NKG2D receptor (WT/Prol/MI6). Means ± SD are shown. Data in J are representative of four experiments.
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fig2: Proliferation is required for RAE-1ε expression on primary fibroblasts. (A) Freshly prepared B6 adult mouse tail fibroblasts were cultured in media supplemented with 2 or 10% serum for ∼3–7 d and harvested for flow cytometry analysis. (B and C) Fibroblasts that had been cultured for >1 mo were serum starved for 1–3 d and processed for staining (B) or RNA analysis (C). Means ± SD are shown. ***, P < 0.0005, unpaired Student’s t test. (D) Samples of serum-starved cells were supplemented with 10% serum and cultured for an additional 3 d. Data in A–D are representative of at least four experiments. (E) Proliferating (Untreated) fibroblasts, or fibroblasts subjected to serum starvation or treatment with LY294002, Roscovitine, or DMSO for 3 d, were stained with 7-AAD and Annexin-V to detect dead and apoptotic cells. (F) Fibroblasts that were serum starved for 3 d were fed with serum for 2 or 3 d in the presence of BrdU to label proliferating cells, before staining the cells intracellularly with BrdU antibodies. (G) Proliferating fibroblasts were serum starved for 3 d before adding 10% serum and BrdU (bottom) or BrdU only (top). After 24 h, cells were stained with BrdU and RAE-1ε antibodies. Gated BrdU+ and BrdU− cells were examined for RAE-1ε expression after excluding 7-AAD+ (dead) cells. (H) Proliferating fibroblasts were stained with RAE-1 antibody, permeabilized, and stained with propidium iodide. Cells in G1, S, or G2 phases of the cell cycle (left) were examined for RAE-1ε expression (right). (I) Freshly prepared tail cells were cultured on fibronectin-coated plates in DMEM supplemented with 10% serum, 100 ng/ml EGF, or no growth factor (−EGF) for 3 d. Cell counts (right, cells/culture) and RAE-1ε staining (left) are shown. Data in E–I are representative of two experiments. (J) Proliferating fibroblasts (Prol) or fibroblasts that were serum starved for 3 d (Starved) were used as target cells for IL-2–activated natural killer cells (LAKs) prepared from NKG2D-deficient (KO) mice or NKG2D WT littermate controls. NKG2D antibody was added to a reaction of WT LAKs with proliferating target cells to block the NKG2D receptor (WT/Prol/MI6). Means ± SD are shown. Data in J are representative of four experiments.

Mentions: As further evidence for a role of proliferation in RAE-1 expression, limiting the concentration of serum and therefore of serum growth factors caused a delay in RAE-1 induction in primary fibroblast cultures (Fig. 2 A). Furthermore, cells that had been cultured for >1 mo in 10% serum to maximally induce RAE-1ε expression rapidly extinguished RAE-1ε cell surface and mRNA expression when deprived of serum (Fig. 2, B and C). Re-addition of 10% serum to the serum-starved cells resulted in restoration of RAE-1ε expression (Fig. 2 D). Serum starvation or cell cycle inhibitor treatment did not cause significant cell death, as very few of the cells were stained with Annexin V or 7-AAD after the treatments (Fig. 2 E). Furthermore, when proliferative conditions were reestablished by restoring serum or washing out inhibitors, most of the cells incorporated BrdU within 2–3 d, demonstrating that the treatments had not caused senescence (Fig. 2 F). Therefore, down-regulation of RAE-1 in these studies was not associated with apoptosis or senescence but rather with the nonproliferating state.


RAE-1 ligands for the NKG2D receptor are regulated by E2F transcription factors, which control cell cycle entry.

Jung H, Hsiung B, Pestal K, Procyk E, Raulet DH - J. Exp. Med. (2012)

Proliferation is required for RAE-1ε expression on primary fibroblasts. (A) Freshly prepared B6 adult mouse tail fibroblasts were cultured in media supplemented with 2 or 10% serum for ∼3–7 d and harvested for flow cytometry analysis. (B and C) Fibroblasts that had been cultured for >1 mo were serum starved for 1–3 d and processed for staining (B) or RNA analysis (C). Means ± SD are shown. ***, P < 0.0005, unpaired Student’s t test. (D) Samples of serum-starved cells were supplemented with 10% serum and cultured for an additional 3 d. Data in A–D are representative of at least four experiments. (E) Proliferating (Untreated) fibroblasts, or fibroblasts subjected to serum starvation or treatment with LY294002, Roscovitine, or DMSO for 3 d, were stained with 7-AAD and Annexin-V to detect dead and apoptotic cells. (F) Fibroblasts that were serum starved for 3 d were fed with serum for 2 or 3 d in the presence of BrdU to label proliferating cells, before staining the cells intracellularly with BrdU antibodies. (G) Proliferating fibroblasts were serum starved for 3 d before adding 10% serum and BrdU (bottom) or BrdU only (top). After 24 h, cells were stained with BrdU and RAE-1ε antibodies. Gated BrdU+ and BrdU− cells were examined for RAE-1ε expression after excluding 7-AAD+ (dead) cells. (H) Proliferating fibroblasts were stained with RAE-1 antibody, permeabilized, and stained with propidium iodide. Cells in G1, S, or G2 phases of the cell cycle (left) were examined for RAE-1ε expression (right). (I) Freshly prepared tail cells were cultured on fibronectin-coated plates in DMEM supplemented with 10% serum, 100 ng/ml EGF, or no growth factor (−EGF) for 3 d. Cell counts (right, cells/culture) and RAE-1ε staining (left) are shown. Data in E–I are representative of two experiments. (J) Proliferating fibroblasts (Prol) or fibroblasts that were serum starved for 3 d (Starved) were used as target cells for IL-2–activated natural killer cells (LAKs) prepared from NKG2D-deficient (KO) mice or NKG2D WT littermate controls. NKG2D antibody was added to a reaction of WT LAKs with proliferating target cells to block the NKG2D receptor (WT/Prol/MI6). Means ± SD are shown. Data in J are representative of four experiments.
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Related In: Results  -  Collection

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Show All Figures
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fig2: Proliferation is required for RAE-1ε expression on primary fibroblasts. (A) Freshly prepared B6 adult mouse tail fibroblasts were cultured in media supplemented with 2 or 10% serum for ∼3–7 d and harvested for flow cytometry analysis. (B and C) Fibroblasts that had been cultured for >1 mo were serum starved for 1–3 d and processed for staining (B) or RNA analysis (C). Means ± SD are shown. ***, P < 0.0005, unpaired Student’s t test. (D) Samples of serum-starved cells were supplemented with 10% serum and cultured for an additional 3 d. Data in A–D are representative of at least four experiments. (E) Proliferating (Untreated) fibroblasts, or fibroblasts subjected to serum starvation or treatment with LY294002, Roscovitine, or DMSO for 3 d, were stained with 7-AAD and Annexin-V to detect dead and apoptotic cells. (F) Fibroblasts that were serum starved for 3 d were fed with serum for 2 or 3 d in the presence of BrdU to label proliferating cells, before staining the cells intracellularly with BrdU antibodies. (G) Proliferating fibroblasts were serum starved for 3 d before adding 10% serum and BrdU (bottom) or BrdU only (top). After 24 h, cells were stained with BrdU and RAE-1ε antibodies. Gated BrdU+ and BrdU− cells were examined for RAE-1ε expression after excluding 7-AAD+ (dead) cells. (H) Proliferating fibroblasts were stained with RAE-1 antibody, permeabilized, and stained with propidium iodide. Cells in G1, S, or G2 phases of the cell cycle (left) were examined for RAE-1ε expression (right). (I) Freshly prepared tail cells were cultured on fibronectin-coated plates in DMEM supplemented with 10% serum, 100 ng/ml EGF, or no growth factor (−EGF) for 3 d. Cell counts (right, cells/culture) and RAE-1ε staining (left) are shown. Data in E–I are representative of two experiments. (J) Proliferating fibroblasts (Prol) or fibroblasts that were serum starved for 3 d (Starved) were used as target cells for IL-2–activated natural killer cells (LAKs) prepared from NKG2D-deficient (KO) mice or NKG2D WT littermate controls. NKG2D antibody was added to a reaction of WT LAKs with proliferating target cells to block the NKG2D receptor (WT/Prol/MI6). Means ± SD are shown. Data in J are representative of four experiments.
Mentions: As further evidence for a role of proliferation in RAE-1 expression, limiting the concentration of serum and therefore of serum growth factors caused a delay in RAE-1 induction in primary fibroblast cultures (Fig. 2 A). Furthermore, cells that had been cultured for >1 mo in 10% serum to maximally induce RAE-1ε expression rapidly extinguished RAE-1ε cell surface and mRNA expression when deprived of serum (Fig. 2, B and C). Re-addition of 10% serum to the serum-starved cells resulted in restoration of RAE-1ε expression (Fig. 2 D). Serum starvation or cell cycle inhibitor treatment did not cause significant cell death, as very few of the cells were stained with Annexin V or 7-AAD after the treatments (Fig. 2 E). Furthermore, when proliferative conditions were reestablished by restoring serum or washing out inhibitors, most of the cells incorporated BrdU within 2–3 d, demonstrating that the treatments had not caused senescence (Fig. 2 F). Therefore, down-regulation of RAE-1 in these studies was not associated with apoptosis or senescence but rather with the nonproliferating state.

Bottom Line: Raet1 genes are directly transcriptionally activated by E2F family transcription factors, which play a central role in regulating cell cycle entry.Induction of RAE-1 occurred in primary cell cultures, embryonic brain cells in vivo, and cells in healing skin wounds and, accordingly, wound healing was delayed in mice lacking NKG2D.Transcriptional activation by E2Fs is likely coordinated with posttranscriptional regulation by other stress responses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cell Biology, 489 Life Sciences Addition, University of California, Berkeley, Berkeley, CA 94720, USA.

ABSTRACT
The NKG2D stimulatory receptor expressed by natural killer cells and T cell subsets recognizes cell surface ligands that are induced on transformed and infected cells and facilitate immune rejection of tumor cells. We demonstrate that expression of retinoic acid early inducible gene 1 (RAE-1) family NKG2D ligands in cancer cell lines and proliferating normal cells is coupled directly to cell cycle regulation. Raet1 genes are directly transcriptionally activated by E2F family transcription factors, which play a central role in regulating cell cycle entry. Induction of RAE-1 occurred in primary cell cultures, embryonic brain cells in vivo, and cells in healing skin wounds and, accordingly, wound healing was delayed in mice lacking NKG2D. Transcriptional activation by E2Fs is likely coordinated with posttranscriptional regulation by other stress responses. These findings suggest that cellular proliferation, as occurs in cancer cells but also other pathological conditions, is a key signal tied to immune reactions mediated by NKG2D-bearing lymphocytes.

Show MeSH
Related in: MedlinePlus