Browsing by Subject "TGF-beta"

The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and homeostasis. BMPs have been shown to pattern the dorsoventral axis in zebrafish (Danio rerio) early during embryonic development by establishing a dorsal-to-­ventral ligand gradient. During tumorigenesis, BMPs primarily function as tumor promoters, as an increase in BMP expression is associated with an increase in invasion, migration, epithelial-­to-­mesenchymal transition (EMT), proliferation and angiogenesis.

Although it is clear that BMPs play multiple roles in these biological events, the precise mechanism by which BMPs mediate these functions is not fully understood. Canonically, BMP ligands signal through cell surface receptor complexes that phosphorylate transcription factors, Smad1, Smad5 and Smad8, which mediate BMP-­ specific gene transcription. While studying BMP signaling during cancer progression, we determined that BMPs unexpectedly signal through the canonical TGF-β-­responsive transcription factors, Smad2 and Smad3.

We determined that BMP-­induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP binding TGF-­β superfamily receptors, ALK3/6, and the Smad2/3 phosphorylating receptors, ALK5/7. BMP signaling through Smad1/5/8 induces MET, while Smad1/5 and Smad2 mediate dorsoventral axis patterning in zebrafish embryos and Smad3 facilitates invasion.

Taken together, our data provides evidence that BMP-­induced Smad2 and Smad3 phosphorylation occurs through a non-­canonical signaling mechanism to mediate multiple biological events. Thus, the signaling mechanisms utilized by BMPs and TGF-­β superfamily receptors are broader than previously appreciated.

The transforming growth factor beta (TGF-beta) signaling pathway is a critical regulator of multiple biological processes that are involved in cancer progression, such as proliferation, migration, invasion and metastasis. TGF-beta ligands bind to multiple high-affinity receptors (TbetaRI, TbetaRII, TbetaRIII), whose expression on the cell surface, and subsequent ability to transduce signaling, can be modulated by ectodomain shedding.

TbetaRIII, also known as betaglycan, is the most abundantly expressed TGF-beta receptor. TbetaRIII suppresses breast cancer progression through inhibiting migration, invasion, metastasis, and angiogenesis. TbetaRIII binds TGF-beta ligands, with membrane-bound TbetaRIII presenting ligand to enhance TGF-beta signaling. However, TbetaRIII can also undergo ectodomain shedding, releasing soluble TbetaRIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound TbetaRIII on TGF-beta signaling and breast cancer biology, here I describe TbetaRIII mutants with impaired (Delta-Shed-TbetaRIII) or enhanced ectodomain shedding (SS-TbetaRIII). Relative to wild-type (WT)-TbetaRIII, Delta-Shed-TbetaRIII increased TGF-beta signaling and blocked TbetaRIII's ability to inhibit breast cancer cell migration and invasion. Conversely, SS-TbetaRIII, which increased soluble TbetaRIII production, decreased TGF-beta signaling and increased TbetaRIII-mediated inhibition of breast cancer cell migration and invasion.

TbetaRI is released from the cell surface by a common sheddase of the A disintegrin and metalloproteinase (ADAM) family, ADAM17. This shedding event results in a downregulation of TGF-beta signaling. Here, I present evidence that a closely related protease, ADAM10, may be a novel sheddase for TbetaRI. A specific ADAM10 inhibitor was able to increase cell surface expression of TbetaRI, and decrease levels of circulating soluble TbetaRI in vivo. Interestingly, inhibition of ADAM10 concurrently increased shedding of TbetaRIII, and was able to alter TGF-beta signaling in a TbetaRIII-dependent manner.

Together, these studies suggest that ectodomain shedding of TGF-beta receptors is an important determinant for regulation of TGF-beta-mediated signaling and biology.

A deficiency in functional pancreatic beta-cells is a defining feature in type 1 and type 2 diabetes. The development of therapeutic strategies for replacement and regeneration of beta-cell mass is a key objective of current diabetes research. The Newgard lab has had a particular focus in exploring novel beta-cell replication pathways in order to identify targets that enhance beta-cell proliferation, survival, and function. The beta-cell developmental transcription factors Nkx6.1 and Pdx-1 each have a profound proliferative effect when overexpressed in young rat islets in vitro. The unique ability of these factors and the pathways that they control to expand functional beta-cell mass while either being neutral or positive for other key functions (survival, insulin secretion) encourages further studies to better elucidate candidate target genes within these pathways.

A major limitation of the research to date is that Nkx6.1 and Pdx-1 only exert their proliferative effects in young (2 months) rodent islets and not in old (8-10 months) rodent islets. Moreover, these factors are only weakly active in human islets, most of which come from middle-aged donors. Nkx6.1 and Pdx-1 engage pathways that are upstream of the core cell cycle machinery and that have the potential to be stimulated in a beta-cell specific manner, but use of this approach will depend on a better understanding of the differences between human and rodent islets, which may be modeled by the differences between old and young rat islets.

The type III TGF-β receptor (TβRIII) is a TGF-β co-receptor that presents ligand to the type II TGF-β receptor to initiate signaling. TβRIII also undergoes ectodomain shedding to release a soluble form (sTβRIII) that can bind ligand, sequestering it away from cell surface receptors. We have previously identified a TβRIII extracellular mutant that has enhanced ectodomain shedding (“super shedding (SS)” – TβRIII-SS). Here we utilize TβRIII-SS to study the balance of cell surface and soluble TβRIII in the context of lung cancer. We demonstrate that expressing TβRIII-SS in lung cancer cell models induces epithelial-mesenchymal transition (EMT) and that these TβRIII-SS (EMT) cells are less migratory, invasive and adhesive and more resistance to gemcitabine. Moreover, TβRIII-SS (EMT) cells exhibit decreased tumorigenicity but increased tumor growth in vitro and in vivo. These studies suggest that the balance of cell surface and soluble TβRIII may regulate a dichotomous role for TβRIII during cancer progression.

We have also demonstrated that cathepsin G and neutrophil elastase, proteases stored in the azurophil granules of neutrophils, can induce cleavage of cell surface TβRIII though no functional sTβRIII was detected. Similarly, activated neutrophils could induce TβRIII shedding on cancer cells. Interestingly, neutrophils generate sTβRIII that is resistant to shedding. These studies suggest that neutrophils may modulate the balance of cell surface and soluble TβRIII in the tumor microenvironment.