To explore the functional role of TIMP-2 in liver, we determined TIMP-2 mRNA levels in primary rat hepatocytes and in total rat liver. Rat hepatocytes constitutively express TIMP-2 mRNA at a low level. Incubation with dexamethasone, prostaglandin E2 and a combination of inflammatory cytokines leads to an up-regulation of TIMP-2 mRNA. In rats in vivo we found a dramatic increase of TIMP-2 expression after intraperitoneal injection of lipopolysaccharide. Compared to our previous findings on TIMP-1 we conclude that TIMP-2 mRNA expression is regulated in a distinct and partially opposite manner. Over-production of TIMP-2 could inhibit the activity of metalloproteinases and thus lead to matrix accumulation. Dysregulation of TIMP-2 synthesis might be involved in the development of liver fibrosis.
Murine tissue inhibitor of metalloproteinases-1 (InTIMP-1) was expressed in haculuvirus-infectcd insect cells (Sf9). The protcin secreted into the culture medium was purified to hornogeneity by meatis of heparin-Sepharose CL-6B and FPLC. The purified protein showed metalloproteiriase-inhibitory activity in two independent assays : revcrse zymography and inhibition of collagcnase activity. Digestion of thc recombinant TIMP-1 with peptide-N-glycanaseF revealed that both N-glycosylation sites are used. 12'1-mTXMP-1 intraveneously injected into a malc Spraguc Dawlcy rat disappeared within 2 min fi-om the circulation. 5 rnin after injection more than 50% of the '"I-mTIMP-1 were found in the liver and 20% in the kidneys. At later times, trichloroacctic-acid-soliible inaterid accumulated in the intestinal tract.Keywords: mctalloprotcinases ; baculovirus; N-glycosylation ; collagcnase; tissue inhibitor of metalloproteinase-I.Proteolytic enzymes are classified by many investigators into four groups : serine-, cysteine-, aspartic-and metalloproteinascs [ 11. Whereas specific protein inhibitors for the protcinases belonging lo the first three groups are well known and have becn studied in detail, inhibitors of mctalloproteinases have only rccently become subject of investigation. The wide-specificity proteinase inhibitor u2-macroglobulin has been described to inhibit metalloproteitiascs [Z]. More specific metalloproteinase inhibitors, however, became known as tissue inhibitors of metalloprcrtcinases (TTMP). Presently three different TIMP have been identified 13-61, The components of the cxtracellular matrix of essentially all cells are degraded by metalloproteinases such as collagenases, stromelysins and gelatinases [ 71. Since the activity of these enzymes is controlled by TIMP, thc balance of nietalloprotei nascs and their inhibitors plays an important role for steady-state concentrations of extracellular matrix components such as collagens and protcoglyoans. Disturbances of this balance due to inl-larnmatory reactions are found in rheumatoid arthritis, ostcoarthritis, fibrosis and cirrhosis [8-121. It has been demonstrated in several laboratories that the in vivo application of lipopolysaccharidc (LPS) leading to the release of inflammatory cytokiiies such as interleukin (IL)-l, tumor-necrosis factor (TNF)a, II,-6 and IL-X results in an increased production of extracellular matrix metalloproteinascs [ 13 I. At the same time, an increase of thc metalloproteinasc inhibitor TIMP-1 has been ob-
a Experiments described herein were supported by the Deutsche Forschungsgemeinschafi Author to whom correspondence should be addressed; Tel: + 49 (241) 808-8830; Fax: (Bonn) and the Fonds der Chemischen Indusaie (Frankfurt). +49 (241) 888-8428. 222
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