Jason C. Mills

We have identified cellular and molecular features of the stem cell niche required for marked amplification of mouse colonic epithelial progenitors (ColEPs) that occurs in response to wounding of the epithelium with dextran sodium sulfate. This regenerative response in areas adjacent to breaches in the epithelial barrier depends on the gut microbiota because ColEP proliferation is markedly diminished in germ-free animals. Analysis of conventionally raised C57BL/6 (B6) knockout mice lacking the Toll-like receptor signal transduction pathway component Myd88 and wild-type animals transplanted with Myd88(-/-) bone marrow, revealed that Myd88-mediated signaling through mesenchymal cells is also required for the ColEP response. Studies of B6 Csf1(op/op) (lacking macrophages) mice, Rag1(-/-) mice, and wild-type mice treated with neutrophil-specific Gr1 mAbs, disclosed that macrophages but not lymphocytes or neutrophils are necessary. GeneChip analysis of laser-capture-microdissected mesenchymal cells coupled with immunohistochemical and electron microscopic studies showed that, during the regenerative response, macrophages in the pericryptal stem cell niche express genes associated with their activation and extend processes to directly contact ColEPs near the crypt base. GeneChip analysis also identified a number of potential molecular mediators of regeneration expressed in the pericryptal progenitor niche, including secreted factors that stimulate epithelial proliferation and proteins involved in extracellular matrix and basement membrane function, stability, and growth factor binding. Together, these studies indicate that the colonic epithelial progenitor niche is a dynamic structure in which macrophages function as mobile "cellular transceivers" that coordinate inputs from luminal microbes and injured epithelium and transmit regenerative signals to neighboring ColEPs.

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.

It has been postulated that the development of amyloid plaques in Alzheimer's disease (AD) may result from an imbalance between the generation and clearance of the amyloid-beta peptide (Abeta). Although familial AD appears to be caused by Abeta overproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of Abeta. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Abeta degradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular Abeta degradation. We found that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded Abeta, lowering levels and producing several fragments after incubation with synthetic human Abeta(1-40) and Abeta(1-42). This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of Abeta were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001) increased brain interstitial fluid Abeta levels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Abeta clearance by promoting Abeta catabolism.