Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF-β1 activation and reduces regulatory ID gene expression

Abstract

Tissue inhibitor of metalloproteinase 1 (TIMP-1) controls matrix metalloproteinase activity through 1:1 stoichiometric binding. Human TIMP-1 fused to a glycosylphosphatidylinositol (GPI) anchor (TIMP-1 -GPI) shifts the activity of TIMP-1 from the extracellular matrix to the cell surface. TIMP-1 -GPI treated renal cell carcinoma cells show increased apoptosis and reduced proliferation. Transcriptomic profiling and regulatory pathway mapping were used to identify the potential mechanisms driving these effects. Significant changes in the DNA binding inhibitors, TGF-β 1/SMAD and BMP pathways resulted from TIMP-1 -GPI treatment. These events were linked to reduced TGF-β 1 signaling mediated by inhibition of proteolytic processing of latent TGF-β 1 by TIMP-1 -GPI.

Pericellular proteolysis in cancer

“…Another strategy to direct TIMP activity to the plasma membrane used constructs in which TIMPs are fused to GPI anchors. Human TIMP-1 fused to a GPI anchor shifted the activity of TIMP-1 from the ECM to the cell surface, which resulted in induction of apoptosis and reduced proliferation of renal cell carcinoma cells (Notohamiprodjo et al 2012). In addition to approaches that aim to improve the selectivity of inhibitors (Cuniasse et al 2005) or neutralizing antibodies (Lund et al 2011) against individual proteases, alternative strategies that target regulatory mechanisms, such as inhibition of NHE1 to prevent acidification of the extracellular environment (Kumar et al 2009;Yang et al 2010) or neutralizing inducers of protease expression such as CD147 (Dean et al 2009), have been proposed.…”

Cell surface engineering using glycosylphosphatidylinositol anchored tissue inhibitor of matrix metalloproteinase‐1 stimulates cutaneous wound healing

“…Specific ELISA was used to assess the production of total vs. active forms of TGF‐β1 by primary dermal fibroblasts in serum‐free media. TIMP‐1‐GPI treatment effectively reduced the levels of active TGF‐β1 relative to the untreated control, suggesting a direct effect on proteolytic processing of the latent TGF‐β1 complex by the surface bound TIMP‐1 (TIMP‐1‐GPI at 7 ng/mL p < 0.0002; 14 ng/mL p < 0.0001) (Figure ).…”

“…Increase in the local concentration of TIMP proteins by the administration of recombinant protein or gene transfer has shown some efficacy in various animal models. Recombinant TIMP‐1‐GPI fusion protein is efficiently incorporated into cell surface membranes, and through this, shiftsTIMP‐1 directly to the cell surface . This modification to the native protein results in enhanced as well as novel biologic effects.…”

“…Wound healing is a complex process linked to the balance of regulatory cytokines/growth factors present. Cell surface engineering using GPI‐anchored TIMP‐1 suppresses processing of growth factors including TGF‐β1 . TGF‐β1 is rapidly induced in response to injury and has some of the broadest effects of any cytokine linked to wound healing .…”

“…We have previously shown that the bioactivity of TIMP‐1 can be altered by changing its cellular distribution, specifically by focusing expression directly on to the cell surface . TIMP‐1 is normally a secreted protein found within the ECM and only loosely associated with the cell surface through its interaction with other proteins.…”