Urokinase is required for the formation of mactinin, an α-actinin fragment that promotes monocyte/macrophage maturation

Sharon D Luikart, Mohammed Masri, Dan Wahl, Tim Hinkel, James M. Beck, Margaret R. Gyetko, Pankaj Gupta, Theodore Oegema

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16 Scopus citations

Abstract

We have previously shown that lysates from HL-60 myeloid leukemia cells or from peripheral blood monocytes are able to degrade α-actinin to form a 31-kDa amino-terminal fragment with monocyte/macrophage maturation promoting activity. In contrast, intact α-actinin, which is a 100-kDa actin-binding protein, has no differentiating activity. The aim of this study was to investigate the enzyme responsible for the degradation of α-actinin to form this fragment, named mactinin. The ability of cell lysates to degrade [125I]α-actinin in the presence of various enzyme inhibitors, including inhibitors of metalloproteinases, cysteine proteinases, and serine proteases, was measured. Phenylmethylsulfonyl fluoride (PMSF) was the only inhibitor able to prevent formation of mactinin by cell lysate degradation of α-actinin, suggesting that a serine protease is responsible for the digestion. Of the various serine proteases tested (thrombin, plasmin, and urokinase), only urokinase was able to produce a 31-kDa band. The urokinase-generated 31-kDa band promoted maturation in HL-60 cells. Amiloride, a specific inhibitor of urokinase, inhibited production of the 31-kDa α-actinin fragment by HL-60 cell lysates. For in vivo tests, inflammatory fluid (from bronchoalvelolar lavage) was collected from uPA (urokinase) knockout mice and their wild-type counterparts after intratracheal challenge with Pneumocystis carinii. Although most (6 of 8) wild-type mice had mactinin in their inflammatory fluid samples, none (0 of 8) of the uPA knockout mice had mactinin present (P<0.01). These results demonstrate that urokinase is necessary and sufficient for the formation of the monocyte/macrophage maturation promoting fragment, mactinin, in vitro and in vivo. These findings support the role of urokinase in the regulation of monocyte/macrophage functions, such as that occurring in inflammatory reactions.

Original languageEnglish (US)
Pages (from-to)99-107
Number of pages9
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1591
Issue number1-3
DOIs
StatePublished - Aug 19 2002

Bibliographical note

Funding Information:
This work was supported by the Department of Veterans Affairs Merit Research grants (SDL and MRG) and National Institutes of Health grants HL 60620 (MRG), HL57011, and HL59823 (JMB).

Keywords

  • Actinin
  • Monocyte
  • Urokinase

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