Role of the matrixin mmp-2 in the multicellular organization of
adipocytes cultured in basement membrane components.
Brown, Lynn M., Heather L. Fox, Stacy A. Hazen, Kathryn F. Lanoue,
Stephen R. Rannels, and Christopher J. Lynch.
Department of Cellular and Molecular Physiology, The Pennsylvania
State University, College of Medicine, Hershey, PA 17033
APStracts 3:0361C, 1996.
Primary rat adipocytes cultured in gels of basement membrane component
gels migrated and organized into three-dimensional, multicellular
clusters. Gross morphological changes seen by phase and fluorescent
microscopy during the organization of these cells are reported,
including the formation of cellular extensions and cell-cell
contacts. Echistatin, a disintegrin, partially inhibited the
formation of multicellular clusters in a concentration-dependent
fashion (IC50 nearly equal to 10nM). In contrast, adipocyte cluster
formation was accelerated in cultures from 7-8 week-old rats as
compared to 15 week-old rats. Multicellular cluster formation was
also accelerated when insulin was added to cultures of adipocytes
from 15 week-old rats but had no significant effect younger animals.
The original extracellular matrix was initially remodeled and
eventually destroyed during the formation of the multicellular
clusters implying that one or more matrix-degrading protease(s) were
being secreted. Adipocyte-conditioned media from serum-starved
cultures were analyzed by zymography and found to contain a divalent
cation-sensitive gelatinase activity at either 72 kDa, 62 kDa or
both. The gelatinase activity bound to gelatin-sepharose-4B in the
presence of high salt and could be eluted by adding 7.5% DMSO to the
high salt buffer, a procedure used to purify matrix metalloproteinase
2 (MMP-2, a 72 kDa matrixin with a 62 kDa mature form). The DMSO
eluant from these columns contained MMP-2 immunoreactivity. MMP-2
concentration and activity was greater in conditioned media from
young compared to older animals, however insulin did not affect the
amount of MMP-2 in adipocyte-conditioned media. The matrixin
inhibitor, 1,10-phenanthroline, not only blocked gelatinase activity
in zymograms, but also prevented extracellular matrix remodeling and
destruction as well as adipocyte migration and the formation of cell
-cell contacts in adipocyte cultures. These observations are
consistent with the hypothesis that the matrixin, MMP-2, is secreted
by adipocytes. While matrixin activity alone may not be sufficient
for the formation of multicellular clusters, the data indicate that
it may have a requisite role in this process.
Received 21 August 1996; accepted in final form 6 September 1996.
APS Manuscript Number C280-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996