Partial purification and characterization of 'injurin-like' factor which stimulates production of hepatocyte growth factor.
We have previously reported the evidence for presence of a humoral factor 'injurin', which induces expression of the hepatocyte growth factor (HGF) gene in MRC-5 human embryonic lung fibroblasts. We have now purified a factor from porcine liver which stimulates HGF production but differs from injurin. When injurin activity was measured as a stimulatory effect on HGF production by MRC-5 cells, this activity was found in various acid extracts from porcine tissues, including liver, kidney, brain, and lung, and acid extracts from the liver was used for purification. When the acid extract was applied to Q-Sepharose anion-exchange chromatography, 50-60% of the total injurin activity was absorbed to the column and the remaining activity was detected in the flow through fractions. Injurin activity was eluted from the Q-Sepharose column by NaCl concentration gradient with four peaks at 0.5-0.6 M, 0.7-0.8 M, 0.9-1.2 M. 1.5-2.0 M NaCl, thereby suggesting that the factor exists in heterogenous or various forms in tissues. The major active fractions were combined and applied to Mono-Q FPLC anion-exchange chromatography. Injurin activity eluted with a single peak at 0.9-1.5 M NaCl and this activity was 4286 fold purified from the starting extract. Addition of this fraction to MRC-5 cells increased the amount of HGF pulse-labeled with [35S]methionine to a 3-4-fold higher level than that seen in control cells, whereas it had no significant effect on HGF mRNA levels. Therefore, this factor seems to stimulate HGF synthesis affecting translational processes and is distinct from the previously characterized injurin which stimulates HGF gene expression. Chemical treatments and SDS-polyacrylamide gel electrophoresis of this injurin-like factor indicated that injurin-like factor is a acid- and heat-stable non-proteinous factor with an apparent M(r) of 8-15 kDa. Since the injurin activity of the factor was decreased by heparinase treatment, the factor may be a polysulfated glycosaminoglycan related to heparin or to heparan sulfate. These results suggest that HGF production may be regulated by this non-proteinous injurin-like factor and that this factor may also play an important role in the regeneration of organs, through translationally enhancing HGF production.
Okazaki
H
H
Division of Biochemistry, Osaka University School of Medicine, Japan.
Matsumoto
K
K
Nakamura
T
T
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D016207:Cytokines / Q000302:isolation & purification* / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D051379:Mice; D009928:Organ Specificity; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D013552:Swine; D014020:Tissue Extracts / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D016207:Cytokines; D012333:RNA, Messenger; D014020:Tissue Extracts; C074248:injurin; D017228:Hepatocyte Growth Factor