Characterization of a novel metalloproteinase (MMP) and a tissue inhibitor of metalloproteinases (TIMP) in tissue repair, inflammation and cancer

Matrix metalloproteinases (MMPs) comprise a family of more than 20 enzymes that take part in the degradation of extracellular matrix and basement membranes during cell migration, angiogenesis and proteolytic activation of growth factors. These events are needed in fetal development and in normal tissue remodelling as well as in wound repair, inflammation and tumour invasion. Our group has recently cloned a new member of the MMP family, human matrix metalloproteinase-21 (MMP-21), which has been suggested to play an important role in embryogenesis and tumour progression. It is expressed in a variety of fetal and adult normal tissues, as well as in some tumour tissues and tumour cell lines, including breast, ovarian, lung, prostate and skin cancers, and osteosarcomas. Not much is known about the regulation of this gene, but it has been shown that transforming growth factor (TGF)-beta1 induces transcription of MMP-21 in the HaCaT keratinocyte cell line, and downregulates it in fibroblasts. Further, phorbol myristate acetate (PMA) and retinoic acid (RA) upregulate the transcription of this gene in monocytic cells. Our primary interest is to study the regulatory pathways of MMP-21 gene expression in detail, and the activation and substrates of this gene. We have also generated two cell lines that overexpress MMP-21 to study the effect of MMP-21 protein on cell morphology, differentiation, proliferation, migration, invasion, and apoptosis.

MMPs are regulated transcriptionally by proenzyme activation, and by specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs-1, -2, -3, and –4). Tissue inhibitor or metalloproteinases-4 is the most recently characterized member of the TIMP family. It is 51% identical to TIMPs-2 and –3, expressed in particular by the placenta, brain, heart and platelets and can induce apoptotic cell death in transformed cells. It inhibits MMPs-1, -2, -3, -7, -9, -14, -26 and TACE. The significance of TIMP-4 in cutaneous biology is still poorly understood. We aim to study the expression of TIMP-4 in vivo in tissue samples from different skin cancers, chronic and acute wounds, and from different chronic skin diseases by immunohistochemistry. In vitro, the expression and regulation of this gene will be studied in monocytes (U937 and THP-1 cells) and T-cells (Jurkat cells). We will also test the effect of different cytokines and growth factors on the expression of TIMP-4 in these cells.

Our project thus aims at further characterization of the biological function and regulation of two novel proteins that may play an important part in cancer biology and inflammation of several different tissues. Methods we will use are cell culturing containing different types of tests for cell proliferation, migration, invasion, and apoptosis, transient and stable transfections, RT-PCR, TaqMan, Western, and immunohistochemistry.