Disruption of Transforming Growth Factor β Signaling by a Novel Ligand-dependent Mechanism

Abstract

Transforming growth factor (TGF)-β is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-β receptors and one or more isoforms of TGF-β, thus the synthesis and secretion of TGF-β as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-β results in TGF-β ligand–receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-β1 in the PCT binds to intracellular TGF-β type II receptor (TβRII). Disruption of the expression of TGF-β1 by antisense TGF-β1 mRNA restores localization of TβRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TβRII (dnTβRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TβRII. Analysis of TGF-β receptor–activated Smad2 suggests the intracellular ligand–receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-β–receptor complex, and define a novel mechanism for modulating the TGF-β signaling pathway.

The role of bone morphogenetic proteins in myeloma cell survival

“…[40] This could be explained by disturbed localization or trafficking of TGF-β receptors. [63] Another explanation may be that the cyclin dependent kinases (CDK) CDK2 and CDK4 can phosphorylate SMAD2/3, thereby inhibiting transactivation of genes. [64] In primary bone marrow myeloma cells SMAD2 was phosphorylated by CDK2 and the transcriptional regulation by TGF-β was disrupted.…”

CDK2 Phosphorylation of Smad2 Disrupts TGF-β Transcriptional Regulation in Resistant Primary Bone Marrow Myeloma Cells

“…As a control, TGF-␤ was secreted (ϳ0.5 ng/10 6 cells/24 h) by mouse PCT lines (7.2 and 16.1) derived from PCT induced by raf/myc-containing retroviruses (32) (Fig. 1A) at the level of pristane-induced PCTs (20). TGF-␤, therefore, was not produced in BM myeloma cells and HMCLs characterized.…”

“…However, the mechanism by which myeloma cells develop TGF-␤ resistance remains unknown. In mice, pristane-induced plasmacytomas (PCTs) present in the lymph nodes are refractory to TGF-␤ due to a lack of functional cell surface TGF-␤ receptors as a consequence of improper receptor trafficking or processing (20,21). Whether a similar mechanism accounts for TGF-␤ resistance in human BM myeloma cells is unclear.…”

“…The absence of functional surface TGF-␤ receptors is the basis for TGF-␤ resistance in several PCT cell lines derived from pristane-induced tumors (20,21). To determine whether the same mechanism underlies TGF-␤ resistance in human myeloma cells, we characterized Smad activation in response to TGF-␤.…”

Transient Inhibition of Endogenous Transforming Growth Factor- β1 (TGFβ1) in Hematopoietic Stem Cells Accelerates Engraftment and Enhances Multi-Lineage Repopulating Efficiency

“…Interestingly, TGFβ1-PMO treatment of LTR-HSC could induce 1-2 cell divisions within 16 hr, in contrast to TGFβ-MAB neutralizing antibodies where cell division was not observed, even after many days in culture (Table 3). Other antisense studies have shown reduced intracellular TGF-β/Receptor complexes [39]. Endogenous TGFβ expression in lin-Sca-1+ cells from TGFβ1 knockout mice (tgfβ1-/-) indicated no detectable TGFβ1 or TGFβ2, and intracellular staining of wild-type lin--Sca-1+ cells showed that endogenous TGFβ expression is predominantly TGFβ1 (Ruscetti, data not shown).…”