MMP28 is constitutively expressed by epithelial cells in many tissues, including the respiratory epithelium in the lung and keratinocytes in the skin. This constitutive expression suggests that MMP28 may serve a role in epithelial cell homeostasis. In an effort to determine its function in epithelial cell biology, we generated cell lines expressing wild-type or catalytically-inactive mutant MMP28 in two pulmonary epithelial cell lines, A549 and BEAS-2B. We observed that over-expression of MMP28 provided protection against apoptosis induced by either serum-deprivation or treatment with a protein kinase inhibitor, staurosporine. Furthermore, we observed increased caspase-3/7 activity in influenza-infected lungs from Mmp28-/- mice compared to wild-type mice, and this activity localized to the airway epithelium but was not associated with a change in viral load. Thus, we have identified a novel role of MMP28 in promoting epithelial cell survival in the lung.

Matrix metalloproteinases are maintained in an inactive state by a bond between the thiol of a conserved cysteine in the prodomain and a zinc atom in the catalytic domain. Once this bond is disrupted, MMPs become active proteinases and can act on a variety of extracellular protein substrates. In vivo, matrilysin (MMP7) activates pro-alpha-defensins (procryptdins), but in vitro, processing of these peptides is slow, with about 50% conversion in 8-12 h. Similarly, autolytic activation of promatrilysin in vitro can take up to 12-24 h for 50% conversion. These inefficient reactions suggest that natural co-factors enhance the activation and activity of matrilysin. We determined that highly sulfated glycosaminoglycans (GAG), such as heparin, chondroitin-4,6-sulfate (CS-E), and dermatan sulfate, markedly enhanced (>50-fold) the intermolecular autolytic activation of promatrilysin and the activity of fully active matrilysin to cleave specific physiologic substrates. In contrast, heparan sulfate and less sulfated forms of chondroitin sulfate did not augment matrilysin activation or activity. Chondroitin-2,6-sulfate (CS-D) also did not enhance matrilysin activity, suggesting that the presentation of sulfates is more important than the overall degree of sulfation. Surface plasmon resonance demonstrated that promatrilysin bound heparin (KD = 400 nM) and CS-E (KD = 630 nM). Active matrilysin bound heparin (KD = 150 nM) but less so to CS-E (KD = 60 muM). Neither form bound heparan sulfate. These observations demonstrate that sulfated GAGs regulate matrilysin activation and its activity against specific substrates.

Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 to -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between LCR 5'HS1 and the epsilon-globin gene was transcriptionally silenced in adult beta-YAC transgenic mice, but deletion of the 352 bp region restored expression. This fragment reduced reporter gene expression in K562 cells and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1 and Mi2 were recruited to the (A)gamma-globin -566 or the (G)gamma-globin -567 GATA sites when gamma-globin expression was low (day 18), but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.