Heike Dahmen

Inhibition of gp130 Signaling in Breast Cancer Blocks Constitutive Activation of Stat3 and Inhibits in Vivo Malignancy

The cytokine receptor gp130 is the common signaling subunit of receptors used by the interleukin (IL)-6 cytokine family. gp130 is widely expressed in breast cancer cell lines and primary tumors. The role of gp130 in breast cancer in vivo is unknown. To study the effect of gp130 inhibition in breast cancer, endogenous gp130 signaling in breast cancer cell lines was blocked with a dominant-negative gp130 protein (DN gp130). DN gp130 inhibited constitutive Stat3 activation in breast cancer cells. Both gp130 and epidermal growth factor receptor (EGFR) have been implicated in constitutive Stat3 activation in breast cancer. There are known physical and functional interactions between gp130 and EGFR. Consistent with this, we show that DN gp130 inhibits signaling downstream of the EGFR in breast cancer cells. The effect of DN gp130 on breast cancer in vivo was assessed with an orthotopic nude mouse model. DN gp130 MDA-231 cells had markedly decreased engraftment, size, and metastasis compared with control cells. These results are particularly striking considering that DN gp130-expressing breast cancer cells grow faster in vitro. We hypothesized that DN gp130 expression results in inhibition of invasion and metastasis in vivo. Marked angiogenesis was present in tumors from control animals and was absent in tumors from DN gp130 animals. We additionally show that tissue inhibitor of metalloproteinase-3, an inhibitor of tumor invasion and angiogenesis, is up-regulated in both MDA-231 DN gp130 cells and tumors. These results, in light of the availability of several potential pharmacological inhibitors of gp130, suggest novel approaches to breast cancer therapy.

The Cytoplasmic Tyrosine Motifs in Full-Length Glycoprotein 130 Have Different Roles in IL-6 Signal Transduction

The function of the signal-transducing receptor subunit glycoprotein 130 (gp130) in the IL-6-receptor complex has previously been studied using carboxyl-terminal deletion mutants or a truncated molecule of ∼60 membrane-proximal amino acids (containing box 1 and box 2) linked to the individual gp130 tyrosine motifs. However, the redundancy of the tyrosine motifs within the cytoplasmic part of gp130 has been neglected. Here we describe the analysis of the function of the individual cytoplasmic tyrosine residues of gp130 in the context of the full-length receptor protein in IL-6 signaling as measured by STAT activation, acute phase protein induction, and stimulation of proliferation. Add-back receptor mutants containing only one cytoplasmic tyrosine have been generated and tested for their efficiency in IL-6 signal transduction. Our studies revealed that tyrosine motifs which have been described to recruit STAT proteins are not equivalent with respect to their potential to activate STAT factors and acute phase protein gene promoters: the two distal tyrosines, Tyr905 and Tyr915, of gp130 were more potent than Tyr767 and Tyr814. Surprisingly, Tyr905 and Tyr915 mediate acute phase protein gene promoter activation stronger than the wild-type receptor containing all six cytoplasmic tyrosine residues. In contrast, Ba/F3 cells stably transfected with add-back receptors containing Tyr767 or Tyr905 were more sensitive to IL-6-induced proliferation than cells expressing the other add-back receptor mutants. Thus, the tyrosine residues in the cytoplasmic part of gp130 were found to contribute differentially to IL-6 signal transduction in the full- length gp130 protein.

A fusion protein of interleukin-11 and soluble interleukin-11 receptor acts as a superagonist on cells expressing gp130

Interleukin‐11 is a hematopoietic cytokine that signals via the signal transducer gp130. Although gp130 is ubiquitously expressed, interleukine‐11 responsiveness is restricted to cells that express the interleukine‐11 receptor α‐subunit. The interleukine‐11 receptor α‐subunit can be functionally replaced by its soluble form indicating that the transmembrane and cytoplasmic parts are not required for signal transduction. Here, we show that a recombinant fusion protein of a fragment of the human interleukine‐11 receptor α‐subunit ectodomain linked to human interleukine‐11 acts as a superagonist on cells expressing gp130 but lacking the membrane‐bound interleukine‐11 receptor α‐subunit. It induces acute phase protein synthesis in hepatoma cells and efficiently promotes proliferation of Ba/F3 cells stably, transfected with gp130. In these bioassays, the fusion protein of a fragment of the human interleukine‐11 receptor α‐subunit ectodomain linked to human interleukine‐11 is 50 times more potent than the combination of interleukine‐11 and the soluble interleukine‐11 receptor α‐subunit. Thus, our findings support the concept that covalent fusion of two soluble proteins required for receptor activation dramatically increases their bioactivity.

Importance of the Membrane-Proximal Extracellular Domains for Activation of the Signal Transducer Glycoprotein 130

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. The gp130 extracellular part is predicted to consist of six individual domains. Whereas the role of the three membrane-distal domains (D1–D3) in binding of IL-6 and IL-11 is well established, the function of the membrane-proximal domains (D4–D6) is unclear. Mapping of a neutralizing mAb to the membrane-proximal part of gp130 suggests a functional role of D4–D6 in receptor activation. Individual deletion of these three domains differentially interferes with ligand binding of the soluble and membrane-bound receptors. All deletion mutants do not signal in response to IL-6 and IL-11. The deletion mutants Δ4 and, to a lesser extent, Δ6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant Δ5 does not respond. Because membrane-bound Δ5 binds IL-6/soluble IL-6R as does wild-type gp130, but does not transduce a signal in response to various stimuli, this domain plays a prominent role in coupling of ligand binding and signal transduction. Replacement of the fifth domain of gp130 by the corresponding domain of the homologous G-CSF receptor leads to constitutive activation of the chimera upon overexpression in COS-7 cells. In HepG2 cells this mutant responds to IL-6 comparable to wild-type gp130. Our findings suggest a functional role of the membrane-proximal domains of gp130 in receptor activation. Thus, within the hematopoietic receptor family the mechanism of receptor activation critically depends on the architecture of the receptor ectodomain.

Constitutive internalization and association with adaptor protein‐2 of the interleukin‐6 signal transducer gp130

The transmembrane protein gp130 is the common signalling receptor subunit for the interleukin‐6 (IL‐6)‐type cytokines. It has recently been shown that the cytoplasmic domain of gp130 contains a dileucine internalization motif and that endocytosis of gp130 occurs signal‐independent. Here, we have studied whether gp130 itself undergoes constitutive internalization or whether its endocytosis is stimulated by formation of the IL‐6/IL‐6R/gp130 complex. Using two different assays, we found that gp130 is internalized independent from IL‐6/IL‐6R stimulation. In addition, we show that gp130 is constitutively associated with the cell surface adaptor complex AP‐2. Our findings strongly suggest endocytosis of gp130 to be constitutive.

Reconstitution of two isoforms of the human interleukin-11 receptor and comparison of their functional properties

Long‐term stable Ba/F3 transfectants (B13Rα1 and B13Rα2) expressing two isoforms of the human IL‐11Rα receptor (α1 full length or α2 lacking the cytoplasmic domain) in combination with human gp130 were established. IL‐11Rα1 and IL‐11Rα2 were each expressed and detected as three bands upon Western blot analysis, with apparent molecular masses in agreement with those of the polypeptide backbone (47 and 44 kDa, respectively) with no, one or two N‐linked sugars. B13Rα1 and B13Rα2 bound IL‐11–thioredoxin with similar efficiencies and proliferated with superimposable dose–response curves to IL‐11, demonstrating that the intracellular domain of IL‐11Rα has no significant contribution on ligand binding and signaling. Analysis of a set of anti‐human gp130 mAbs confirmed the similar responsiveness of B13Rα1 and B13Rα2 transfectants.