Stat3 Activates the Receptor Tyrosine Kinase Like Orphan Receptor-1 Gene in Chronic Lymphocytic Leukemia Cells

Ping Li,David Harris,Zeev Estrov,Zhiming Liu,Jie Liu,Michael Keating,Mikhail V Blagosklonny

doi:10.1371/journal.pone.0011859

Ping Li, David Harris + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0011859

Copy DOI

Journal: PloS one	Publication Date: Jul 29, 2010
Citations: 116	License type: CC BY 4.0

Affiliation: The University of Texas MD Anderson Cancer Center

Abstract

BackgroundThe receptor tyrosine kinase like orphan receptor (ROR)-1 gene is overexpressed in chronic lymphocytic leukemia (CLL). Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors γ-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1.Methodology/Principal FindingsBecause IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells.Conclusion/SignificanceOur data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.

Highlights

Receptor tyrosine kinase like orphan (Ror) proteins are type-I transmembrane receptor tyrosine kinase members of the neurotrophic tyrosine kinase receptor superfamily that regulate the developmental wingless-type (Wnt) signaling pathway [1]
Unstimulated MM1 cells express low to undetectable levels of phosphotyrosine-Stat3, and exposure of MM1 cells to IL-6 induces Stat3 phosphorylation [16,17]
Other investigators have reported that the Wnt signaling pathway is activated [22] and the Wnt receptor ROR1 gene is overexpressed in chronic lymphocytic leukemia (CLL) cells

Summary

Introduction

Receptor tyrosine kinase like orphan (Ror) proteins are type-I transmembrane receptor tyrosine kinase members of the neurotrophic tyrosine kinase receptor superfamily that regulate the developmental wingless-type (Wnt) signaling pathway [1]. Ror proteins possess a tyrosine kinase domain and a proline-rich domain straddled by two Ser/ Thr-rich domains [2]. Ror protein functions are involved in skeletal development. Human ROR2 mutations cause well-characterized skeletal defects such as dominant brachydactily type B, a condition of shortened or missing digits [3,4], and recessive Robinow syndrome, a form of short-limbed dwarfism [5,6]. The receptor tyrosine kinase like orphan receptor (ROR)-1 gene is overexpressed in chronic lymphocytic leukemia (CLL). Because Stat is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors c-interferon activation sequence-like elements typically activated by Stat, we hypothesized that Stat activates ROR1

Methods

Results

Conclusion