Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Defining epithelial cell progenitors in the human oxyntic mucosa.

In the human stomach, the oxyntic epithelium includes numerous tubular invaginations consisting of short pits opening into long glands. The pit is lined by pit cells, whereas the gland is composed of three regions: the base, containing zymogenic cells; the neck, containing neck cells; and the isthmus, composed of little known immature cells and of parietal cells, which are also scattered through the neck and base. The aim of this study was to examine the ultrastructure of the immature cells and to determine their relation to mature cells. To do so, normal oxyntic mucosal biopsies from subjects ranging from 20-43 years old were fixed in aldehydes and postfixed in reduced osmium for electron microscopy and morphometric analysis. The immature cells were sorted out into four classes, whose roles were clarified by comparison with the thoroughly investigated mouse oxyntic epithelium. The first class was composed of the least differentiated immature cells, which were rare and characterized by minute, dense, or cored secretory granules and were accordingly named mini-granule cells. Their function was not clarified. The second class consisted of pre-pit cells, which were characterized by few dense mucous granules and give rise to pit cells that ascend the pit wall and, after reaching the luminal surface, die or are extruded. Both pre-pit and pit cells underwent continuous renewal and, therefore, together constituted a renewal system referred to as pit cell lineage. The third class, or pre-neck cells, characterized by cored secretory granules, give rise to neck cells that descend toward the base region and differentiate further into pre-zymogenic cells, which finally become zymogenic cells. The latter eventually degenerate and die. Thus pre-neck cells and their progeny constitute a renewing system, designated zymogenic cell lineage. The fourth class, or pre-parietal cells, characterized by long microvilli and few tubulovesicles, differentiate into parietal cells that descend along the neck and base regions and eventually degenerate and die. Pre-parietal and parietal cells represent a renewing system referred to as parietal cell lineage. While the origin of the last three classes of progenitor cells has not been elucidated, it is likely that they arise either from an unidentified multipotential stem cell, possibly the mini-granule cell itself, or from the mitotic activity of pre-pit and pre-neck cells. In conclusion, the human oxyntic epithelium is composed of continually renewing cells organized in distinct cell lineages.