Leif R. Lund

Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N‐terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280‐289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C‐terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross‐linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

Plasminogen activator inhibitors: hormonally regulated serpins

Etude de la regulation hormonale des enzymes appartenant a la famille de serine protease inhibiteur (plasminogen activator et urokinase)

CANCER INVASION AND TISSUE REMODELING : COMMON THEMES IN PROTEOLYTIC MATRIX DEGRADATION

Analysis of extracellular matrix degradation systems has led to the insight that in cancer invasion there is often crucial interplay between cancer cells and several types of surrounding non-neoplastic stromal cells. Likewise, in normal tissue remodeling processes, the synthesis of proteolytic components is often distributed between several cell types, and there are strong similarities between neoplastic and non-neoplastic processes in the same tissue. Thus, tissue remodeling events are excellent models for studies of extracellular proteolysis in cancer. This has become even clearer by recent analyses of genetically modified mice.

Plasminogen activation and cancer

Breakdown of the extracellular matrix is crucial for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases. The activity of each of these proteases is regulated by an array of activators, inhibitors and cellular receptors. Thus, the generation of plasmin involves the pro-enzyme plasminogen, the urokinase type plasminogen activator uPA and its pro-enzyme pro-uPA, the uPA inhibitor PAI-1, the cell surface uPA receptor uPAR, and the plasmin inhibitor alpha(2)-antiplasmin. Furthermore, the regulation of extracellular proteolysis in cancer involves a complex interplay between cancer cells and non-malignant stromal cells in the expression of the molecular components involved. For some types of cancer, this cellular interplay mimics that observed in the tissue of origin during non-neoplastic tissue remodelling processes. We propose that cancer invasion can be considered as uncontrolled tissue remodelling. Inhibition of extracellular proteases is an attractive approach to cancer therapy. Because proteases have many different functions in the normal organism, efficient inhibition will have toxic side effects. In cancer invasion, like in normal tissue remodelling processes, there appears to be a functional overlap between different extracellular proteases. This redundancy means that combinations of protease inhibitors must be used. Such combination therapy, however, is also likely to increase toxicity. Therefore for each type of cancer, a combination of protease inhibitors that is optimised with respect to both maximal therapeutic effect and minimal toxic side effects need to be identified.

Cancer invasion and tissue remodeling : cooperation of protease systems and cell types

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non‐neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI‐1 and MMPs) can be expressed by either the non‐neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non‐neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Functional overlap between two classes of matrix-degrading proteases in wound healing.

Retarded wound healing was found in mice deficient in the serine protease precursor plasminogen, as well as in wild‐type mice treated with the metalloprotease inhibitor galardin, but in both cases wound closure was ultimately completed in all mice within 60 days. The expression of several matrix metalloproteases in keratinocytes migrating to cover the wound was strongly enhanced by galardin treatment. However, when plasminogen‐deficient mice were treated with galardin, healing was completely arrested and wound closure was not seen during an observation period of 100 days, demonstrating that protease activity is essential for skin wound healing. The requirement for both plasminogen deficiency and metalloprotease inhibition for complete inhibition of the healing process indicates that there is a functional overlap between the two classes of matrix‐degrading proteases, probably in the dissection of the fibrin‐rich provisional matrix by migrating keratinocytes. Each class alone is capable of maintaining sufficient keratinocyte migration to regenerate the epidermal surface, although this function would normally be performed by both classes acting in parallel. Since there are strong similarities between the proteolytic mechanisms in wound healing and cancer invasion, these results predict that complete arrest of this latter process in therapeutic settings will require the use of inhibitors of both classes of proteases.

Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis

During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

Transforming growth factor-beta is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts.

We have studied the mechanism of a transforming growth factor‐beta (TGF‐beta)‐stimulated production of type‐1 plasminogen activator inhibitor (PAI‐1) in WI‐38 human lung fibroblasts. TGF‐beta causes an early increase in the PAI‐1 mRNA level which reaches a maximal 50‐fold enhancement after 8 h. Blocking of protein synthesis with cycloheximide causes an equally strong increase in the level of PAI‐1 mRNA. Quantitative studies of the effect of TGF‐beta on PAI‐1 protein levels in cell extracts and culture media by using monoclonal antibodies are consistent with the effect on PAI‐1 mRNA. The results suggest a primary effect of TGF‐beta on PAI‐1 gene transcription, and also suggest the possibility that the transcription of this gene in non‐induced cells may be suppressed by a short‐lived negatively regulating protein. Urokinase‐type (u‐PA) and tissue‐type (t‐PA) plasminogen activators are decreased in the culture media of TGF‐beta‐treated cells concomitantly with the increase in PAI‐1 accumulation. These findings show that a primary and important biological effect of TGF‐beta may be an overall decreased extracellular proteolytic activity, and give an insight into the molecular mechanisms underlying TGF‐beta action at the genetic level.

Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17

Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4+ T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.

Impaired Wound Contraction in Stromelysin-1–Deficient Mice

OBJECTIVE To determine whether the deletion of stromelysin-1, a single metalloproteinase gene product, will alter the time course and quality of dermal wound repair in mice. SUMMARY BACKGROUND DATA After dermal injury, a highly coordinated program of events is initiated by formation of a fibrin clot, followed by migration of keratinocytes, contraction of the dermis, recruitment of inflammatory macrophages, formation of granulation tissue with angiogenesis, and finally tissue remodeling. Matrix metalloproteinases are rapidly induced in the dermis and granulation tissue and at the leading edge of the epidermis in the healing wounds. METHODS Incisional and circular full-thickness wounds 2 to 10 mm were made in the dermis of stromelysin-1-deficient and wild-type mice. The wounds were analyzed for rate of cellular migration and epithelialization. The wound contraction was examined by immunohistochemical staining for alpha-smooth muscle actin and fluorescent staining for fibrillar actin. RESULTS Independent of the age of the animal, excisional wounds in stromelysin-1-deficient mice failed to contract and healed more slowly than those in wild-type mice. Cellular migration and epithelialization were unaffected in the stromelysin-1-deficient animals. The functional defect in these mice is failure of contraction during the first phase of healing because of inadequate organization of actin-rich stromal fibroblasts. CONCLUSIONS Excisional dermal wound healing is impaired in mice with a targeted deletion in the stromelysin-1 gene. Incisional wound healing is not affected. These data implicate stromelysin-1 proteolysis during early wound contraction and indicate that stromelysin-1 is crucial for the organization of a multicellular actin network.