ABSTRACT
Murine tissue inhibitor of metalloproteinases-1 (mTIMP-1) was expressed in baculovirus-infected insect cells (Sf9). The protein secreted into the culture medium was purified to homogeneity by means of heparin-Sepharose CL-6B and FPLC. The purified protein showed metalloproteinase-inhibitory activity in two independent assays: reverse zymography and inhibition of collagenase activity. Digestion of the recombinant TIMP-1 with peptide-N-glycanaseF revealed that both N-glycosylation sites are used. 125I-mTIMP-1 intraveneously injected into a male Sprague Dawley rat disappeared within 2 min from the circulation. 5 min after injection more than 50% of the 125I-mTIMP-1 were found in the liver and 20% in the kidneys. At later times, trichloroacetic-acid-soluble material accumulated in the intestinal tract.
Subject(s)
Glycoproteins/isolation & purification , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/isolation & purification , Amino Acid Sequence , Animals , Baculoviridae/genetics , Cells, Cultured , Glycoproteins/biosynthesis , Glycosylation , Male , Metabolic Clearance Rate , Mice , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Spodoptera , Tissue Distribution , Tissue Inhibitor of MetalloproteinasesSubject(s)
Antigens, CD , Interleukin-6/physiology , Receptors, Interleukin/physiology , Trans-Activators , Amino Acid Sequence , Animals , Base Sequence , Consensus Sequence , Cytokine Receptor gp130 , DNA-Binding Proteins/physiology , Endocytosis , Gene Expression Regulation , Haptoglobins/genetics , Humans , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Phosphotyrosine , Promoter Regions, Genetic , RNA, Messenger/genetics , Receptors, Interleukin/chemistry , Receptors, Interleukin-6 , STAT3 Transcription Factor , Signal Transduction , Solubility , Transcription Factors/physiology , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
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.
