Date of Award
Doctor of Philosophy
Genetics, Development and Cell Biology
The aim of this project is to examine the role of proteases and protease inhibitors in the regulation of cell proliferation. It also describes a simple, high yield purification of bovine platelet derived TGF-[beta] that uses a novel bioassay to specifically identify TGF-[beta] mediated activity;The composition of the extracellular matrix (ECM) is an important determinant of the rate cellular proliferation and differentiation. Growth factors and other proteins with growth regulatory activities can affect the composition of the ECM. Growth regulators are also associated with the ECM and can be released by the action of ECM associated proteolysis;We have examined the effects of plasmin on mammalian cell proliferation. We have found that plasmin inhibits the proliferation of CCL64 (mink lung epithelial), R1B-CCL64 (TGF-[beta]-receptor mutants of CCL64), BSC-1 (green monkey kidney epithelial) and AKR-2B (mouse fibroblast) cell lines. Plasmin inhibits DNA synthesis in a dose dependent manner with 75-100% inhibition at 50 mU/ml as measured by [superscript]3H-thymidine incorporation and labeling indices. The plasmin inhibition is abrogated by the serine protease inhibitors aprotinin and PMSF indicating proteolysis is required for inhibition. Plasmin releases a soluble growth inhibitory factor from the ECM. The inhibitor, termed matrixin-A, is not inhibited by aprotinin or PMSF. Matrixin has been purified, has a molecular weight of 22 KDa, does not inhibit protein synthesis and is not TGF-[beta]. Plasmin does not release TGF-[beta] from the extra-cellular matrix even from cells known to secrete large quantities of TGF-[beta]. Plasmin treated ECM inhibits DNA synthesis in the TGF-[beta] receptor mutants, R1B-CCl 64, and fails to induce PAI-1 in TGF-[beta] responsive cells. Plasmin may inhibit mammalian cell proliferation by releasing matrixin from the ECM;Type-1 transforming growth factor-[beta] has been purified to homogeneity from bovine platelets by using a high yield, two step procedure. TGF-[beta] mediated growth inhibitory activity was assayed using a novel approach in which both TGF-[beta] responding cells (CCL64) and non-responding cells, TGF-[beta]-receptor mutants (R1B-CCL64) were used in a [superscript]3H-thymidine incorporation assay. The assay specifically identifies TGF-[beta] mediated inhibition from other potential growth inhibitory activities in platelets. The rapid purification procedure includes non-denaturing gel filtration followed by reverse phase HPLC of acid/ethanol extracted bovine platelets. Yields of 10 [mu]g of TGF-[beta]1 per gram of platelets and specific activities of 1.2 x 10[superscript]8 units/ug were obtained.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Scott William Miller
Miller, Scott William, "The characterization and purification of matrixin-A, a plasmin releasable growth inhibitor associated with the extracellular matrix " (1992). Retrospective Theses and Dissertations. 9806.