Limits...
Novel mechanism of action on Hedgehog signaling by a suppressor of fused carboxy terminal variant.

Tostar U, Finta C, Rahman MF, Shimokawa T, Zaphiropoulos PG - PLoS ONE (2012)

Bottom Line: Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL.Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes PTCH1 and HHIP.Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.

ABSTRACT
The Suppressor of Fused (SUFU) protein plays an essential role in the Hedgehog (HH) signaling pathway, by regulation of the GLI transcription factors. Two major isoforms of human SUFU are known, a full-length (SUFU-FL) and a carboxy-terminal truncated (SUFU- ΔC) variant. Even though SUFU- ΔC is expressed at an equivalent level as SUFU-FL in certain tissues, the function of SUFU-ΔC and its impact on HH signal transduction is still unclear. In two cell lines from rhabdomyosarcoma, a tumor type associated with deregulated HH signaling, SUFU-ΔC mRNA was expressed at comparable levels as SUFU-FL mRNA, but at the protein level only low amounts of SUFU-ΔC were detectable. Heterologous expression provided support to the notion that the SUFU-ΔC protein is less stable compared to SUFU-FL. Despite this, biochemical analysis revealed that SUFU-ΔC could repress GLI2 and GLI1ΔN, but not GLI1FL, transcriptional activity to the same extent as SUFU-FL. Moreover, under conditions of activated HH signaling SUFU-ΔC was more effective than SUFU-FL in inhibiting GLI1ΔN. Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL. Additionally, confocal microscopy revealed a co-localization of GLI1FL with SUFU-ΔC but not SUFU-FL in aggregate structures. Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes PTCH1 and HHIP. Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.

Show MeSH

Related in: MedlinePlus

Dose-dependent SUFU repression of GLI1 activity in constitutively active Ptch1−/− MEFs.Different amounts of Myc-tagged SUFU-FL or SUFU-ΔC expression constructs were co-transfected with 50 ng GLI1 expression constructs (Panel A, GLI1FL; panel B, GLI1ΔN), 12xGLIBS-luc and pRL-SV reporter plasmids, and the luciferase activity was measured. Error bars indicate the standard deviation. Note the increased capacity of SUFU-ΔC relative to SUFU-FL in inhibiting GLI1ΔN transcriptional activity.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3362617&req=5

pone-0037761-g005: Dose-dependent SUFU repression of GLI1 activity in constitutively active Ptch1−/− MEFs.Different amounts of Myc-tagged SUFU-FL or SUFU-ΔC expression constructs were co-transfected with 50 ng GLI1 expression constructs (Panel A, GLI1FL; panel B, GLI1ΔN), 12xGLIBS-luc and pRL-SV reporter plasmids, and the luciferase activity was measured. Error bars indicate the standard deviation. Note the increased capacity of SUFU-ΔC relative to SUFU-FL in inhibiting GLI1ΔN transcriptional activity.

Mentions: We also investigated whether HH signaling activation may modulate the SUFU repressive effects on GLI1. For this purpose mouse embryonic fibroblast cells lacking Ptch1 (Ptch1−/− MEFs), and therefore characterized by a constitutively active HH signaling pathway, were used. The results indicated that the maximal repression was observed with SUFU-FL acting on GLI1FL, as seen with NIH3T3 cells. SUFU-ΔC, on the other hand, was found to be more effective than SUFU-FL in repressing the GLI1ΔN variant (Fig 5A and B).


Novel mechanism of action on Hedgehog signaling by a suppressor of fused carboxy terminal variant.

Tostar U, Finta C, Rahman MF, Shimokawa T, Zaphiropoulos PG - PLoS ONE (2012)

Dose-dependent SUFU repression of GLI1 activity in constitutively active Ptch1−/− MEFs.Different amounts of Myc-tagged SUFU-FL or SUFU-ΔC expression constructs were co-transfected with 50 ng GLI1 expression constructs (Panel A, GLI1FL; panel B, GLI1ΔN), 12xGLIBS-luc and pRL-SV reporter plasmids, and the luciferase activity was measured. Error bars indicate the standard deviation. Note the increased capacity of SUFU-ΔC relative to SUFU-FL in inhibiting GLI1ΔN transcriptional activity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362617&req=5

pone-0037761-g005: Dose-dependent SUFU repression of GLI1 activity in constitutively active Ptch1−/− MEFs.Different amounts of Myc-tagged SUFU-FL or SUFU-ΔC expression constructs were co-transfected with 50 ng GLI1 expression constructs (Panel A, GLI1FL; panel B, GLI1ΔN), 12xGLIBS-luc and pRL-SV reporter plasmids, and the luciferase activity was measured. Error bars indicate the standard deviation. Note the increased capacity of SUFU-ΔC relative to SUFU-FL in inhibiting GLI1ΔN transcriptional activity.
Mentions: We also investigated whether HH signaling activation may modulate the SUFU repressive effects on GLI1. For this purpose mouse embryonic fibroblast cells lacking Ptch1 (Ptch1−/− MEFs), and therefore characterized by a constitutively active HH signaling pathway, were used. The results indicated that the maximal repression was observed with SUFU-FL acting on GLI1FL, as seen with NIH3T3 cells. SUFU-ΔC, on the other hand, was found to be more effective than SUFU-FL in repressing the GLI1ΔN variant (Fig 5A and B).

Bottom Line: Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL.Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes PTCH1 and HHIP.Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.

ABSTRACT
The Suppressor of Fused (SUFU) protein plays an essential role in the Hedgehog (HH) signaling pathway, by regulation of the GLI transcription factors. Two major isoforms of human SUFU are known, a full-length (SUFU-FL) and a carboxy-terminal truncated (SUFU- ΔC) variant. Even though SUFU- ΔC is expressed at an equivalent level as SUFU-FL in certain tissues, the function of SUFU-ΔC and its impact on HH signal transduction is still unclear. In two cell lines from rhabdomyosarcoma, a tumor type associated with deregulated HH signaling, SUFU-ΔC mRNA was expressed at comparable levels as SUFU-FL mRNA, but at the protein level only low amounts of SUFU-ΔC were detectable. Heterologous expression provided support to the notion that the SUFU-ΔC protein is less stable compared to SUFU-FL. Despite this, biochemical analysis revealed that SUFU-ΔC could repress GLI2 and GLI1ΔN, but not GLI1FL, transcriptional activity to the same extent as SUFU-FL. Moreover, under conditions of activated HH signaling SUFU-ΔC was more effective than SUFU-FL in inhibiting GLI1ΔN. Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL. Additionally, confocal microscopy revealed a co-localization of GLI1FL with SUFU-ΔC but not SUFU-FL in aggregate structures. Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes PTCH1 and HHIP. Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.

Show MeSH
Related in: MedlinePlus