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Suppression of latent transforming growth factor (TGF)-beta1 restores growth inhibitory TGF-beta signaling through microRNAs.

Dogar AM, Towbin H, Hall J - J. Biol. Chem. (2011)

Bottom Line: A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression.The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-β1 processing factors, thrombospondin-1, and furin, respectively.For cells with high levels of latent TGF-β, this provides a potentially widespread mechanism of escape from TGF-β-mediated growth arrest at the earliest point in the signaling pathway, TGF-β processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.

ABSTRACT
Cancer cells secreting excess latent TGF-β are often resistant to TGF-β induced growth inhibition. We observed that RNAi against TGF-β1 led to apoptotic death in such cell lines with features that were, paradoxically, reminiscent of TGF-β signaling activity and that included transiently enhanced SMAD2 and AKT phosphorylation. A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression. The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-β1 processing factors, thrombospondin-1, and furin, respectively. Our data suggest a novel mechanism in which latent TGF-β1, thrombospondin 1, and furin form a microRNA-mediated regulatory feedback loop. For cells with high levels of latent TGF-β, this provides a potentially widespread mechanism of escape from TGF-β-mediated growth arrest at the earliest point in the signaling pathway, TGF-β processing.

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Related in: MedlinePlus

Increased maturation of TGF-β upon down-regulation                                        of latent TGF-β1 in HeLa cells involves                                        miRNAs.                                    A, HeLa cells transfected with                                        THBS1 plasmid. Total RNA was isolated 24 h                                    post-transfection and subjected to Q-PCR using                                        THBS1 specific primers. B,                                    HeLa cells transfected with THBS1 plasmid.                                    Caspase 3/7 activity was measured from supernatants 24 h                                    post-transfection (left panel). Caspase 3/7                                    activity was measured from supernatants of recipient HeLa cells                                    48 h post-transfer (right panel) (mean of                                    triplicate transfections ±S.D.). C and                                        D, cells transfected with                                        THBS1 and FURIN                                    3′-UTR reporter plasmids were treated after 24 h with                                    siRNAs or miRNAs. Luciferase activity was measured 48 h after                                    plasmid transfections. Relative luciferase activity is displayed                                    (mean of triplicate transfections ± S.D.).                                        E and F, cells grown in                                    media containing 1% FBS were treated with miRNAs. Western                                    blots of proteins from lysates, and supernatants are displayed.                                        G and H, HeLa cells were                                    transfected with miR-18a and miR-24. Total RNA was isolated 72 h                                    post-transfection and Q-PCR analysis was performed. Relative                                    expressions of TGF-β1 and FURIN mRNAs                                    are displayed (mean of PCR triplicates; single RNA samples                                    ± S.D.). I, HeLa cells were                                    simultaneously transfected with 15 nm siTGFβ1                                    and increasing doses of mir-18a, mir-24, siCon, and miR-181b.                                    Western blot analyses were performed 24 h post-transfection.
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Figure 4: Increased maturation of TGF-β upon down-regulation of latent TGF-β1 in HeLa cells involves miRNAs. A, HeLa cells transfected with THBS1 plasmid. Total RNA was isolated 24 h post-transfection and subjected to Q-PCR using THBS1 specific primers. B, HeLa cells transfected with THBS1 plasmid. Caspase 3/7 activity was measured from supernatants 24 h post-transfection (left panel). Caspase 3/7 activity was measured from supernatants of recipient HeLa cells 48 h post-transfer (right panel) (mean of triplicate transfections ±S.D.). C and D, cells transfected with THBS1 and FURIN 3′-UTR reporter plasmids were treated after 24 h with siRNAs or miRNAs. Luciferase activity was measured 48 h after plasmid transfections. Relative luciferase activity is displayed (mean of triplicate transfections ± S.D.). E and F, cells grown in media containing 1% FBS were treated with miRNAs. Western blots of proteins from lysates, and supernatants are displayed. G and H, HeLa cells were transfected with miR-18a and miR-24. Total RNA was isolated 72 h post-transfection and Q-PCR analysis was performed. Relative expressions of TGF-β1 and FURIN mRNAs are displayed (mean of PCR triplicates; single RNA samples ± S.D.). I, HeLa cells were simultaneously transfected with 15 nm siTGFβ1 and increasing doses of mir-18a, mir-24, siCon, and miR-181b. Western blot analyses were performed 24 h post-transfection.

Mentions: We searched next for the source of elevated TGF-β processing activity. TGF-β stimulates transcription of many of its own activators including FURIN and THBS1, a member of the secreted thrombospondin family (46). Soluble TSP1 binds to and activates latent TGF-β in cell supernatants as well as in a cell-free system (47). FURIN is a protease, which nicks latent TGF-β primarily in the cell but there are also reports of its secretion (48). The mRNA levels of THBS1 and FURIN were slightly elevated after TGF-β1 RNAi (Fig. 2A), however both proteins showed a strong induction on siTGFβ1 treatment more in accordance with a post-transcriptional activation (Fig. 3D). Transfection of cells with a cDNA expressing THBS1 (Fig. 4A) was associated with a 3-fold increase in caspase 3/7 activity 2–3 days post-transfection, and a 5–6 fold increase in recipient HeLa cells on media transfer (Fig. 4B). Recently, THBS1 was shown to be targeted by miRNAs of the miR-17∼92 cluster (49), which is itself transcriptionally repressed by p53 (50). In one account miR-18a was unveiled as a major regulator of tumor angiogenesis via its interaction with THBS1 (18). Recently, miR-18a was shown to regulate SMAD2 (31). The relatively long 3′-UTR of FURIN shows conserved predicted binding sites for miR-17∼92, miR-137, and miR-24 (www.targetscan.org: V5.1), all of which were repressed by TGF-β1 RNAi (Fig. 2C). MiR-24 is one of the most highly expressed miRNAs in HeLa cells and is reportedly suppressed by Smad signaling (35). Furthermore, expression of miR-24 was reported to be altered by TGF-β in hepatocellularcarcinoma cells (51), although functions of miR-24 in TGF-β signaling are likely cell-type specific (32). To determine whether THBS1 or FURIN were post-transcriptionally up-regulated by siTGFβ1 we used luciferase reporter constructs bearing their full-length 3′-UTRs. In contrast to siCon, increasing doses of siTGFβ1 elevated luciferase activity suggesting that the induction of these factors during TGF-β RNAi derived to a significant degree from derepression of their UTR (Fig. 4, C and D). To establish whether miR-18a and miR-24 contributed to the regulation of TSP1 and FURIN during TGF-β1 RNAi, we co-transfected miRNA mimics and their respective reporter constructs into cells. MiR-18a and miR-24 dose-dependently inhibited luciferase-THBS1 and luciferase-FURIN by upto 50% (Fig. 4, C and D). To confirm that miR-18a and miR-24 are able to regulate endogenous TSP1 and FURIN, respectively, we isolated protein from cells treated independently with miRNA mimics. A strong reduction in TSP1 was observed 24 h after treatment with miR-18a (Fig. 4E), whereas no inhibition was obtained from miR-24 (Fig. 4F). In contrast, FURIN was very strongly repressed by miR-24, at both mRNA (Fig. 4H) and protein levels (Fig. 4F). Moreover, basal levels of latent un-nicked TGF-β1 in cell lysates rose as levels of extracellular TSP1 and cellular FURIN dropped on addition of miR-18a and miR-24, respectively, but not on treatment with miR-20b (data not shown). In the case of miR-18a mimic, this resulted at least partly from increased transcription of TGF-β1 (Fig. 4G), whereas for miR-24, it was likely due to intracellular accumulation of the un-nicked latent TGF-β1 as FURIN was repressed. To confirm the functional importance of the repression of miR-18a and miR-24 during the siTGF-β1-mediated processing of latent TGF-β, we again co-transfected cells with each mimic in combination with a 15 nm dose of siTGF-β1. Similar to the TGF-β1 overexpression vector, increasing amounts of either miR-18a or miR-24 countered the effects of siTGF-β1: levels of latent TGF-β1 protein were raised (Fig. 4I) and caspase 3/7 activity was attenuated (Fig. 2, D and E). No such effects were obtained from siCon and only a minor effect was observed at the highest dose from addition of miR-181b (Fig. 4I).


Suppression of latent transforming growth factor (TGF)-beta1 restores growth inhibitory TGF-beta signaling through microRNAs.

Dogar AM, Towbin H, Hall J - J. Biol. Chem. (2011)

Increased maturation of TGF-β upon down-regulation                                        of latent TGF-β1 in HeLa cells involves                                        miRNAs.                                    A, HeLa cells transfected with                                        THBS1 plasmid. Total RNA was isolated 24 h                                    post-transfection and subjected to Q-PCR using                                        THBS1 specific primers. B,                                    HeLa cells transfected with THBS1 plasmid.                                    Caspase 3/7 activity was measured from supernatants 24 h                                    post-transfection (left panel). Caspase 3/7                                    activity was measured from supernatants of recipient HeLa cells                                    48 h post-transfer (right panel) (mean of                                    triplicate transfections ±S.D.). C and                                        D, cells transfected with                                        THBS1 and FURIN                                    3′-UTR reporter plasmids were treated after 24 h with                                    siRNAs or miRNAs. Luciferase activity was measured 48 h after                                    plasmid transfections. Relative luciferase activity is displayed                                    (mean of triplicate transfections ± S.D.).                                        E and F, cells grown in                                    media containing 1% FBS were treated with miRNAs. Western                                    blots of proteins from lysates, and supernatants are displayed.                                        G and H, HeLa cells were                                    transfected with miR-18a and miR-24. Total RNA was isolated 72 h                                    post-transfection and Q-PCR analysis was performed. Relative                                    expressions of TGF-β1 and FURIN mRNAs                                    are displayed (mean of PCR triplicates; single RNA samples                                    ± S.D.). I, HeLa cells were                                    simultaneously transfected with 15 nm siTGFβ1                                    and increasing doses of mir-18a, mir-24, siCon, and miR-181b.                                    Western blot analyses were performed 24 h post-transfection.
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Figure 4: Increased maturation of TGF-β upon down-regulation of latent TGF-β1 in HeLa cells involves miRNAs. A, HeLa cells transfected with THBS1 plasmid. Total RNA was isolated 24 h post-transfection and subjected to Q-PCR using THBS1 specific primers. B, HeLa cells transfected with THBS1 plasmid. Caspase 3/7 activity was measured from supernatants 24 h post-transfection (left panel). Caspase 3/7 activity was measured from supernatants of recipient HeLa cells 48 h post-transfer (right panel) (mean of triplicate transfections ±S.D.). C and D, cells transfected with THBS1 and FURIN 3′-UTR reporter plasmids were treated after 24 h with siRNAs or miRNAs. Luciferase activity was measured 48 h after plasmid transfections. Relative luciferase activity is displayed (mean of triplicate transfections ± S.D.). E and F, cells grown in media containing 1% FBS were treated with miRNAs. Western blots of proteins from lysates, and supernatants are displayed. G and H, HeLa cells were transfected with miR-18a and miR-24. Total RNA was isolated 72 h post-transfection and Q-PCR analysis was performed. Relative expressions of TGF-β1 and FURIN mRNAs are displayed (mean of PCR triplicates; single RNA samples ± S.D.). I, HeLa cells were simultaneously transfected with 15 nm siTGFβ1 and increasing doses of mir-18a, mir-24, siCon, and miR-181b. Western blot analyses were performed 24 h post-transfection.
Mentions: We searched next for the source of elevated TGF-β processing activity. TGF-β stimulates transcription of many of its own activators including FURIN and THBS1, a member of the secreted thrombospondin family (46). Soluble TSP1 binds to and activates latent TGF-β in cell supernatants as well as in a cell-free system (47). FURIN is a protease, which nicks latent TGF-β primarily in the cell but there are also reports of its secretion (48). The mRNA levels of THBS1 and FURIN were slightly elevated after TGF-β1 RNAi (Fig. 2A), however both proteins showed a strong induction on siTGFβ1 treatment more in accordance with a post-transcriptional activation (Fig. 3D). Transfection of cells with a cDNA expressing THBS1 (Fig. 4A) was associated with a 3-fold increase in caspase 3/7 activity 2–3 days post-transfection, and a 5–6 fold increase in recipient HeLa cells on media transfer (Fig. 4B). Recently, THBS1 was shown to be targeted by miRNAs of the miR-17∼92 cluster (49), which is itself transcriptionally repressed by p53 (50). In one account miR-18a was unveiled as a major regulator of tumor angiogenesis via its interaction with THBS1 (18). Recently, miR-18a was shown to regulate SMAD2 (31). The relatively long 3′-UTR of FURIN shows conserved predicted binding sites for miR-17∼92, miR-137, and miR-24 (www.targetscan.org: V5.1), all of which were repressed by TGF-β1 RNAi (Fig. 2C). MiR-24 is one of the most highly expressed miRNAs in HeLa cells and is reportedly suppressed by Smad signaling (35). Furthermore, expression of miR-24 was reported to be altered by TGF-β in hepatocellularcarcinoma cells (51), although functions of miR-24 in TGF-β signaling are likely cell-type specific (32). To determine whether THBS1 or FURIN were post-transcriptionally up-regulated by siTGFβ1 we used luciferase reporter constructs bearing their full-length 3′-UTRs. In contrast to siCon, increasing doses of siTGFβ1 elevated luciferase activity suggesting that the induction of these factors during TGF-β RNAi derived to a significant degree from derepression of their UTR (Fig. 4, C and D). To establish whether miR-18a and miR-24 contributed to the regulation of TSP1 and FURIN during TGF-β1 RNAi, we co-transfected miRNA mimics and their respective reporter constructs into cells. MiR-18a and miR-24 dose-dependently inhibited luciferase-THBS1 and luciferase-FURIN by upto 50% (Fig. 4, C and D). To confirm that miR-18a and miR-24 are able to regulate endogenous TSP1 and FURIN, respectively, we isolated protein from cells treated independently with miRNA mimics. A strong reduction in TSP1 was observed 24 h after treatment with miR-18a (Fig. 4E), whereas no inhibition was obtained from miR-24 (Fig. 4F). In contrast, FURIN was very strongly repressed by miR-24, at both mRNA (Fig. 4H) and protein levels (Fig. 4F). Moreover, basal levels of latent un-nicked TGF-β1 in cell lysates rose as levels of extracellular TSP1 and cellular FURIN dropped on addition of miR-18a and miR-24, respectively, but not on treatment with miR-20b (data not shown). In the case of miR-18a mimic, this resulted at least partly from increased transcription of TGF-β1 (Fig. 4G), whereas for miR-24, it was likely due to intracellular accumulation of the un-nicked latent TGF-β1 as FURIN was repressed. To confirm the functional importance of the repression of miR-18a and miR-24 during the siTGF-β1-mediated processing of latent TGF-β, we again co-transfected cells with each mimic in combination with a 15 nm dose of siTGF-β1. Similar to the TGF-β1 overexpression vector, increasing amounts of either miR-18a or miR-24 countered the effects of siTGF-β1: levels of latent TGF-β1 protein were raised (Fig. 4I) and caspase 3/7 activity was attenuated (Fig. 2, D and E). No such effects were obtained from siCon and only a minor effect was observed at the highest dose from addition of miR-181b (Fig. 4I).

Bottom Line: A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression.The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-β1 processing factors, thrombospondin-1, and furin, respectively.For cells with high levels of latent TGF-β, this provides a potentially widespread mechanism of escape from TGF-β-mediated growth arrest at the earliest point in the signaling pathway, TGF-β processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.

ABSTRACT
Cancer cells secreting excess latent TGF-β are often resistant to TGF-β induced growth inhibition. We observed that RNAi against TGF-β1 led to apoptotic death in such cell lines with features that were, paradoxically, reminiscent of TGF-β signaling activity and that included transiently enhanced SMAD2 and AKT phosphorylation. A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression. The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-β1 processing factors, thrombospondin-1, and furin, respectively. Our data suggest a novel mechanism in which latent TGF-β1, thrombospondin 1, and furin form a microRNA-mediated regulatory feedback loop. For cells with high levels of latent TGF-β, this provides a potentially widespread mechanism of escape from TGF-β-mediated growth arrest at the earliest point in the signaling pathway, TGF-β processing.

Show MeSH
Related in: MedlinePlus