Generation of Globular Fragment of Adiponectin by Leukocyte Elastase Secreted by Monocytic Cell Line THP-1

Abstract

Previous studies revealed that carboxyl-terminal fragment containing the globular domain of adiponectin exists in human plasma. Although it is proposed that the globular fragment is generated by proteolytic cleavage, the place and responsible enzyme of the cleavage are still unclear. In this study, we evaluated the activity to cleave adiponectin in culture medium of several cell lines in vitro. Adiponectin cleavage into several carboxyl-terminal fragments containing the globular domain was observed in the medium of phorbol 12-myristate 13-acetate-stimulated monocytic cell lines THP-1 and U937. The molecular masses of the major products were 25, 20, and 18 kDa. The cleavage was thought to be mediated by leukocyte elastase (also known as neutrophil elastase) based on the following observations. First, the cleavage was inhibited by serine-protease inhibitors [phenylmethylsulfonylfluoride, Pefabloc SC (Roche Diagnostics, Basel, Switzerland) and aprotinin] and by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK. Second, no activity was detected after THP-1 cells had fully differentiated into macrophages. Third, purified leukocyte elastase cleaved adiponectin with the same cleavage pattern as THP-1 cells. Finally, leukocyte elastase secreted by activated neutrophils cleaved adiponectin into the globular fragments. Amino-terminal sequence analysis revealed that cleavage sites of adiponectin by purified leukocyte elastase were between 38Thr and 39Cys, 40Ala and 41Gly, 44Ala and 45Gly, 91Ala and 92Glu, and 110Ala and 111Ala (the numbering of the positions of the amino acids starts at the signal sequence), suggesting that the cleavage occurs in the collagenous domain. These data indicate that the cleavage of adiponectin by leukocyte elastase secreted from activated monocytes and/or neutrophils could be a candidate for the mechanism of the generation of the globular fragment of adiponectin.

Adiponectin as a tissue regenerating hormone: more than a metabolic function

“…A much-debated point is how and in what conditions gAd is produced, since the modalities of its production are still unclear. waki et al [16] reported that fAd can be cleaved by leukocyte elastase secreted from activated monocytes and/or neutrophils. This mechanism could be a possible source of the generation of gAd in the plasma.…”

“…Adiponectin is secreted in "full-length" (fAd) form, which can be cleaved into smaller "globular" (gAd) form by the elastase secreted by activated monocytes and/or neutrophils [15,16]. Monomers of fAd can assemble forming three different multimers: low molecular weight (LMw) trimers, middle molecular weight (MMw) hexamers, and high molecular weight (HMw) 12-18 multimers [17,18].…”

Globular adiponectin, acting via adiponectin receptor-1, inhibits leptin-stimulated oesophageal adenocarcinoma cell proliferation

“…A truncated form containing only the C-terminal portion (globular adiponectin, gAdipo) is also produced by proteolytic cleavage and is biologically active, although the relative serum levels of the 2 forms in different disease states have not been investigated to any great extent and fAdipo may be cleaved within inflamed tissues to gAdipo by elastase (Waki et al 2006).…”

Hormonal regulation of immune modulators in human breast tissue

“…Several studies have shown the negative effect of adiponectin on the proliferation of several cancer cell lines (144,146). Epidemiological studies, using serum adiponectin level as a measure of tissue exposure, suggested that adiponectin was a protective factor against breast cancer (147)(148)(149)(150)(151). In vitro studies have demonstrated that adiponectin induced apoptosis and inhibited migration and invasion of breast cancer cells (152,153).…”