Epidermal growth factor and mitogenic proteinases
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. A serine proteinase of Mr 68K from three cultured fibroblasts, namely, the human embryonic lung fibroblasts, the human foetal skin fibroblasts and mouse L-fibroblasts was purified by antibody affinity chromotography. The anti body was raised against a 80K leukocyte membrane proteinase. The immunochemically related proteinase was trypsin-like and demonstrated 3.5 fold lower activity towards a elastase -specific substrate. The proteinase was extremely labile; autocatalysis resulted in heterogenous low-Mr-proteins. Trypsinisation of the monolayer resulted in the isolation of the 65K proteinase as well. This was an artefact of the isolation procedure. Harvesting of cells by scraping the monolayer yielded a 71K and 68K protein. Inhibitors of the 68K proteinase include DFP, PMSF, ?-1-AT, Zn2+, and benzamidine. Ca2+ and Mg2+ stimulated its activity. Intact cell monolayers possessed the ability to hydrolyze the chromogenic substrate S2160 showing that this arginine-specific proteinase was on the cell-surface. Addition of APG to these intact cell monolayers inhibited the cell-surface proteinase activity. ?-1-AT was also found in inhibit the same cell-surface proteinase activity as APG. Increasing the APG concentration to as high as 1.5mg/ml did not cause complete inhibition of the enzyme activity. The ability of DFP to completely inhibit this enzyme suggested that 54% of the activity was unaffected by APG and ?-1-AT was due to seric hindrance by these large m acromolecules. Washing the monolayer by 3mM EGTA just prior to assay, permitted 100% inhibition by 0.3mg/ml APG. This suggested that Ca2+ may play a role in protecting 54% of the activity that was normally not accessible to APG. This confirms the previous observation that steric hindrance prevents complete inhibition by APG. Cells of higher passage numbers or had reached confluency possessed a lower degree of proteinase activity. Inhibition of this cell-surface enzyme activity caused cessation of growth of human fibroblasts. This inhibition on growth by APG is reversible and non-toxic. Other proteinase inhibitors such as ?-1-antitrypsin and soybean trypsin inhibitor (SBTI) also can inhibit cell multiplication. SBTI immobilised on agarose beads also inhibited growth. This suggested growth inhibition was brought about on the cell-surface. ?-1-antitrypsin caused partial inhibition of mouse L-cells. Cell-surface protein iodination experiments showed that the 85K - 90K protein was a possible endogenous substrate for the proteinase because it increased in APG-treated cells. Also, a lower amount of low-Mr -proteins was observed in APG-treated cells. These proteins which could be the degradation products of the substrate were lesser in APG-treated cells because the proteolytic action was inhibited by APG. Conditioned media from growing human fibroblasts (strain MP-S) contained a 32K proteinase inhibitor. A 70K proteinase inhibitor was released by confluent cells instead. Iodination of conditioned media from APG-treated cells revealed that a 70K protein was also inhibited. EGF-stimulated mitogenesis was inhibited by APG. The rate of internalisation was slowed down. No qualitative difference was observed in normal and APG-treated cells. APG and ?-1-AT did not affect the binding of 125I-EGF to the receptor. APG also inhibited mitogenesis by a phorbol ester PMA. A cell-surface protein of Mr 55K which was normally absent after incubation with EGF over 4 hours, remained on the cell-surface of APG-treated cells even after 24 hours incubation with EGF. Tentative results suggested that EGF-induced mobilisation of Ca2+ from the endoplasmic recticulum was inhibited in APG-treated cells. The external Ca2+ was important for EGF and phytohaemagglutinin (PHA) mitogenesis. The blockage of Ca2+ entry via the calcium-channel by (+)-verapamil inhibited the mitogenic action of EGF and PHA but not thrombin. The 68K proteinase can be stimulated by Ca2+ in vitro. The presence of external Ca2+ can also stimulate the cell-surface proteinase activity in intact cells. Elevated calcium concentrations reduced inhibition of cell-surface proteinase activity by APG. The higher cell-surface proteinase activity resulted in the reversal of growth inhibition by APG. Experiments with quin-2/AM showed that elevation extracellular calcium to 10mM caused increased free cytoplasmic Ca2+ in both normal and APG-treated cells. This rise in cytoplasmic Ca2+ could exert an independent mitogenic action. However, it is possible that part of the action of extracellular calcium may be due to stimulation of the APG-specific proteinase.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/1985