MT1-MMP controls human mesenchymal stem cell trafficking and differentiation.

Human mesenchymal stem cells (hMSCs) localized to bone marrow, nonhematopoietic organs, as well as perivascular niches are postulated to traffic through type I collagen-rich stromal tissues to first infiltrate sites of tissue damage, inflammation, or neoplasia and then differentiate. Nevertheless, the molecular mechanisms supporting the ability of hMSCs to remodel 3-dimensional (3D) collagenous barriers during trafficking or differentiation remain undefined. Herein, we demonstrate that hMSCs degrade and penetrate type I collagen networks in tandem with the expression of a 5-member set of collagenolytic matrix metalloproteinases (MMPs). Specific silencing of each of these proteases reveals that only a single membrane-tethered metalloenzyme, termed MT1-MMP, plays a required role in hMSC-mediated collagenolysis, 3D invasion, and intravasation. Further, once confined within type I collagen-rich tissue, MT1-MMP also controls hMSC differentiation in a 3D-specific fashion. Together, these data demonstrate that hMSC invasion and differentiation programs fall under the control of the pericellular collagenase, MT1-MMP.

Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1.

The ability of carcinoma cells arising at primary sites to cross their underlying basement membrane (BM), a specialized form of extracellular matrix that subtends all epithelial cells, and to access the host vasculature are central features of the malignant phenotype. The initiation of the invasive phenotype has been linked to the aberrant expression of zinc-finger transcriptional repressors, like Snail1, which act by triggering an epithelial-mesenchymal cell-like transformation (EMT-like) via the regulation of largely undefined, downstream effectors. Herein, we find that Snail1 induces cancer cells to (i) degrade and perforate BM barriers, (ii) initiate angiogenesis, and (iii) and intravasate vascular networks in vivo via a matrix metalloproteinase (MMP)-dependent process. Unexpectedly, the complete Snail1 invasion program can be recapitulated by expressing directly either of the membrane-anchored metalloproteinases, MT1-MMP or MT2-MMP. The pro-invasive, angiogenic, and metastatic activities of MT1-MMP and MT2-MMP are unique relative to all other metalloproteinase family members and cannot be mimicked in vivo by the secreted MMPs, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, or MMP-13. Further, siRNA-specific silencing of MT1-MMP and MT2-MMP ablates completely the ability of Snail1 to drive cancer cell BM invasion, induce angiogenesis, or trigger intravasation. Taken together, these data demonstrate that MT1-MMP and MT2-MMP cooperatively function as direct-acting, pro-invasive factors that confer Snail1-triggered cells with the key activities most frequently linked to morbidity and mortality in cancer.

Serum-free culture of rat postnatal neurons derived from the dorsal motor nucleus of the vagus.

Previous studies on dorsal motor nucleus of the vagus (DMNV) neurons have mainly used in vivo animal models and in vitro brainstem slices. Primary culture of postnatal DMNV neurons in defined serum free medium has not been reported. We report a method for culture of postnatal rat DMNV neurons using serum free medium. Cultured DMNV neurons contain both Hu positive precursor cells and mature cells staining positively for microtubule associated protein 2 (MAP2) and choline acetyltransferase. Exposure of cultured DMNV neurons to glutamate (10(-7) to 10(-3)M) induced an increase in intracellular calcium concentration ([Ca(2+)](i)) in a dose-dependent manner, indicating the functional presence of glutamate receptors. Voltage-dependent calcium currents were present in cultured DMNV neurons. Active cell proliferation was demonstrated by BrdU incorporation. Upon removal of beta FGF, the percentage of MAP2 positive mature neurons was significantly increased from 36+/-3 to 73+/-3%. Our study demonstrates that postnatal rat DMNV neurons cultured in serum free medium retain morphological and physiological characteristics of DMNV neurons in situ.

Activation of c-fos expression in the rat inferior olivary nucleus by ghrelin.

Ghrelin, a novel 28-amino-acid hormone secreted by gastric oxyntic glands, stimulates food intake and induces adiposity. We examined whether ghrelin activates the inferior olivary nucleus. Systemic administration of ghrelin (37 nmol/kg) induced the expression of c-fos immunoreactivity in inferior olive neurons (n=6 rats). The number of neurons containing c-fos staining was significantly increased in the ghrelin-treated rats (65+/-14 vs.11+/-6 positive neurons, n=5). No significant difference in c-fos-positive neurons was observed between left (32+/-5) and right (33+/-6) inferior olivary nuclei. The number of c-fos-positive neurons in rats with bilateral vagotomy was not significantly different from those with intact vagal nerves. The present study demonstrates that ghrelin induces c-fos expression in inferior olivary nucleus via a central mechanism.

Snail1 controls epithelial-mesenchymal lineage commitment in focal adhesion kinase-null embryonic cells.

Mouse embryonic cells isolated from focal adhesion kinase (FAK)-null animals at embryonic day 7.5 display multiple defects in focal adhesion remodeling, microtubule dynamics, mechanotransduction, proliferation, directional motility, and invasion. To date, the ability of FAK to modulate cell function has been ascribed largely to its control of posttranscriptional signaling cascades in this embryonic cell population. In this paper, we demonstrate that FAK unexpectedly exerts control over an epithelial-mesenchymal transition (EMT) program that commits embryonic FAK-null cells to an epithelial status highlighted by the expression of E-cadherin, desmoplakin, and cytokeratins. FAK rescue reestablished the mesenchymal characteristics of FAK-null embryonic cells to generate committed mouse embryonic fibroblasts via an extracellular signal-related kinase- and Akt-dependent signaling cascade that triggered Snail1 gene expression and Snail1 protein stabilization. These findings indentify FAK as a novel regulator of Snail1-dependent EMT in embryonic cells and suggest that multiple defects in FAK(-/-) cell behavior can be attributed to an inappropriate commitment of these cells to an epithelial, rather than fibroblastic, phenotype.

Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs.

Epithelial-mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.

Intercellular calcium waves in cultured enteric glia from neonatal guinea pig.

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura-2 AM microfluorimetry. Single-cell stimulation was performed with a micromanipulator-driven glass pipette. Data were expressed as mean +/- SEM and analyzed by Student's t-test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% +/- 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 microM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 microM) significantly decreased the percentage of cells responding to mechanical stimulation (6% +/- 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% +/- 13% vs. 60% +/- 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles.