Abstract
Melanoma differentiation associated gene-7 (mda-7/IL-24) is a member of the IL-10 family of cytokines, with ubiquitous direct and “bystander” tumor-selective killing properties. MDA-7/IL-24 protein binds distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2, IL-22R1/IL-20R1 and IL-22R1/IL-20R2. Recombinant MDA-7/IL-24 protein induces endogenous mda-7/IL-24 expression in a receptor-dependent manner; since A549 cells that lack a complete set of cognate receptors are not responsive to exogenous protein. The mechanism of MDA-7/IL-24 ligand-receptor biology is not well understood. We explored the interaction of MDA-7/IL-24 with its’ receptors and the consequences of ligand-receptor docking. Using both pharmacological and genetic approaches we demonstrate that MDA-7/IL-24 internalization employs the clathrin-mediated endocytic pathway leading to degradation of receptors via the lysosomal/ubiquitin proteosomal pathway. This clathrin-mediated endocytosis is dynamin-dependent. This study resolves a novel mechanism of MDA-7/IL-24 protein “bystander” function, which involves receptor/protein-mediated internalization and receptor degradation.
Highlights
Using both pharmacological and genetic approaches we demonstrate that MDA-7/ IL-24 internalization employs the clathrin-mediated endocytic pathway leading to degradation of receptors via the lysosomal/ubiquitin proteosomal pathway
The IL-10 gene family member mda-7 known as Interleukin-24 (IL-24) is a well-characterized multifunctional tumor suppressor displaying broadspectrum cancer-specific cell killing activity [1,2,3,4,5,6]. mda-7/IL-24 was first identified and cloned using a differentiation induction subtraction hybridization (DISH) screening approach with metastatic human melanoma cells induced to terminally differentiate by treatment with recombinant human interferon and the protein kinase C activator mezerein [7, 8]. mda-7/IL-24 displays restricted expression, which is evident in melanocytes, peripheral blood leukocytes and a subset of immune cells [5, 6, 9]
DU-145 cells were treated with His-tagged recombinant MDA7/IL-24 and the abundancy of different subunits of the MDA-7/IL-24 receptor on the plasma membrane was examined by surface biotinylation and Western blotting
Summary
The IL-10 gene family member mda-7 (melanoma differentiation associated gene-7) known as Interleukin-24 (IL-24) is a well-characterized multifunctional tumor suppressor displaying broadspectrum cancer-specific cell killing activity [1,2,3,4,5,6]. mda-7/IL-24 was first identified and cloned using a differentiation induction subtraction hybridization (DISH) screening approach with metastatic human melanoma cells induced to terminally differentiate by treatment with recombinant human interferon and the protein kinase C activator mezerein [7, 8]. mda-7/IL-24 displays restricted expression, which is evident in melanocytes, peripheral blood leukocytes and a subset of immune cells [5, 6, 9]. MDA-7/IL-24 induces the expression of the chaperone protein BiP/GRP78, which in turn results in endoplasmic reticulum (ER) stress and cell death [16]. This cytokine regulates toxic autophagy through a miR-221-Beclin-1 axis [17, 18]. Clathrinmediated endocytosis is a well-established method for the internalization of a diverse array of cargo proteins [24] This leads to endosomal processing of both the ligand and the receptor [24], and the receptors are either recycled to the surface or undergo degradation mediated by proteasomes or lysosomes [25]. We uncover a role of the lysosomal/ ubiquitin proteosomal pathway, and Dynamin in this dynamic physiological process
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